Supplementary Figure 3 from Cell Motility in Chronic Lymphocytic Leukemia: Defective Rap1 and αLβ2 Activation by Chemokine

Supplementary Plot 1 from c-Abl Expression in Chronic Lymphocytic Leukemia Cells: Clinical and Therapeutic Implications

B-cell malignancies (BCM) originate from the same cell of origin, but at different maturation stages and have distinct clinical phenotypes. Although genetic risk variants for individual BCMs have been identified, an agnostic, genome-wide search for shared genetic susceptibility has not been performed. We explored genome-wide association studies of chronic lymphocytic leukaemia (CLL, N = 1,842), Hodgkin lymphoma (HL, N = 1,465) and multiple myeloma (MM, N = 3,790). We identified a novel pleiotropic risk locus at 3q22.2 (NCK1, rs11715604, P = 1.60 × 10−9) with opposing effects between CLL (P = 1.97 × 10−8) and HL (P = 3.31 × 10−3). Eight established non-HLA risk loci showed pleiotropic associations. Within the HLA region, Ser37 + Phe37 in HLA-DRB1 (P = 1.84 × 10−12) was associated with increased CLL and HL risk (P = 4.68 × 10−12), and reduced MM risk (P = 1.12 × 10−2), and Gly70 in HLA-DQB1 (P = 3.15 × 10−10) showed opposing effects between CLL (P = 3.52 × 10−3) and HL (P = 3.41 × 10−9). By integrating eQTL, Hi-C and ChIP-seq data, we show that the pleiotropic risk loci are enriched for B-cell regulatory elements, as well as an over-representation of binding of key B-cell transcription factors. These data identify shared biological pathways influencing the development of CLL, HL and MM. The identification of these risk loci furthers our understanding of the aetiological basis of BCMs.

Several chronic lymphocytic leukaemia (CLL) susceptibility loci have been reported; however, much of the heritable risk remains unidentified. Here we perform a meta-analysis of six genome-wide association studies, imputed using a merged reference panel of 1,000 Genomes and UK10K data, totalling 6,200 cases and 17,598 controls after replication. We identify nine risk loci at 1p36.11 (rs34676223, P =5.04 × 10 −13 ), 1q42.13 (rs41271473, P =1.06 × 10 −10 ), 4q24 (rs71597109, P =1.37 × 10 −10 ), 4q35.1 (rs57214277, P =3.69 × 10 −8 ), 6p21.31 (rs3800461, P =1.97 × 10 −8 ), 11q23.2 (rs61904987, P =2.64 × 10 −11 ), 18q21.1 (rs1036935, P =3.27 × 10 −8 ), 19p13.3 (rs7254272, P =4.67 × 10 −8 ) and 22q13.33 (rs140522, P =2.70 × 10 −9 ). These new and established risk loci map to areas of active chromatin and show an over-representation of transcription factor binding for the key determinants of B-cell development and immune response.

Chronic myeloid leukemia (CML) is a malignant disease of the primitive hematologic cell which is driven by BCR-ABL1 tyrosine kinase activity.[1][1] Although in recent years CML treatment has been drastically improved by the tyrosine kinase inhibitor (TKI) imatinib, at least one-third of patients

ALOX5 is implicated in chronic myeloid leukemia development in mouse leukemic stem cells, but its importance in human chronic myeloid leukemia is unknown. Functional ALOX5 was assessed using an LTB4 ELISA and ALOX5, and LTB4R1 mRNA expression was determined via a TaqMan gene expression assay. LTB4R1 and 5-LOX protein levels were assessed by cell surface flow cytometry analysis. At diagnosis ALOX5 was below normal in both blood and CD34(+) stem cells in all patients. On treatment initiation, ALOX5 levels increased in all patients except those who were destined to progress subsequently to blast crisis. LTB4 levels were increased despite low ALOX5 expression, suggesting that the arachidonic acid pathway is functioning normally up to the point of LTB4 production. However, the LTB4 receptor (BLT1) protein in newly diagnosed patients was significantly lower than after a period of treatment (P<0.0001). The low level of LTB4R1 at diagnosis explains the downregulation of ALOX5. In the absence of LTB4R1, the arachidonic acid pathway intermediates (5-HEPTE and LTA4) negatively regulate ALOX5. ALOX5 regulation is aberrant in chronic myeloid leukemia patients and may not be important for the development of the disease. Our data suggest caution when extrapolating mouse model data into human chronic myeloid leukemia.

Richard Houlston and colleagues report results of a genome-wide association study of chronic lymphocytic leukemia. They validate several new susceptibility loci for this disease, including variants near POT1, TERC and TERT. Genome-wide association studies (GWAS) of chronic lymphocytic leukemia (CLL) have shown that common genetic variation contributes to the heritable risk of CLL. To identify additional CLL susceptibility loci, we conducted a GWAS and performed a meta-analysis with a published GWAS totaling 1,739 individuals with CLL (cases) and 5,199 controls with validation in an additional 1,144 cases and 3,151 controls. A combined analysis identified new susceptibility loci mapping to 3q26.2 (rs10936599, P = 1.74 × 10−9), 4q26 (rs6858698, P = 3.07 × 10−9), 6q25.2 (IPCEF1, rs2236256, P = 1.50 × 10−10) and 7q31.33 (POT1, rs17246404, P = 3.40 × 10−8). Additionally, we identified a promising association at 5p15.33 (CLPTM1L, rs31490, P = 1.72 × 10−7) and validated recently reported putative associations at 5p15.33 (TERT, rs10069690, P = 1.12 × 10−10) and 8q22.3 (rs2511714, P = 2.90 × 10−9). These findings provide further insights into the genetic and biological basis of inherited genetic susceptibility to CLL.

Prospective identification of patients whose chronic myeloid leukemia (CML) will progress to blast crisis is currently not possible. PP2A is a phosphatase and tumor suppressor that regulates cell proliferation, differentiation, and survival. Cancerous inhibitor of PP2A (CIP2A) is a recently described inhibitor of PP2A in breast and gastric cancer. The aim of this study was to investigate whether CIP2A played a role in CML and whether PP2A or its inhibitor proteins CIP2A or SET could predict clinical outcome. At the time of diagnosis of CML, patients who will later progress to blast crisis have significantly higher levels of CIP2A protein (P < .0001) than patients who do not progress, suggesting that PP2A is functionally inactive. We show that the potential mechanism for disease progression is via altered phosphorylation of the oncogene c-Myc. Knockdown of CIP2A results in increased PP2A activity, decreased c-Myc levels, and a decrease in BCR-ABL1 tyrosine kinase activity. We demonstrate that CIP2A levels at diagnosis can consistently predict patients who will progress to blast crisis. The data show that CIP2A is biologically and clinically important in CML and may be a novel therapeutic target.

A definitive diagnosis of CML requires either the demonstration of the t(9;22) Philadelphia chromosome translocation (by FISH), or BCR-ABL1 fusion gene (qRT-PCR). Both techniques provide important clinical information, but are usually confined to specialist regional laboratories as they are costly,

Abstract B-cell chronic lymphocytic leukemia (CLL) is the most common lymphoid malignancy in the western world. Although strong evidence for an inherited susceptibility is provided by first-degree relatives of CLL patients having >5-fold increased risk of themselves developing CLL, the genetic basis of CLL predisposition is principally unknown. Low penetrance risk alleles for CLL have been identified at 11q24.1 (rs735665, GRAMD1B), 15q23 (rs7176508), 2q37.1 (rs13397985, SP140), 2q37.3 (rs757978, FARP2), 2q13 (rs17483466, ACOXL) and 6p25.3 (rs872071, IRF4). The strongest evidence for association was for a single nucleotide polymorphism (rs872071) in the 3′ UTR of the gene encoding the interferon regulatory factor-4 (IRF4) transcription factor. Given a role in determining risk of disease it is plausible that these allelic variants also play a role in disease progression and overall survival. To test this hypothesis, polymorphic status was determined in a large case series of 403 patients diagnosed with CLL recruited via 5 UK clinical centers, and correlated with established prognostic markers and clinical outcome. Variant IRF4 (defined by rs872071) predicts for a more aggressive disease course; carriers of the disease-associated allele at the IRF4 locus (G/G and G/A genotypes) had a significantly shorter treatment-free survival (TFS) compared to non-carriers (A/A genotype). The risk allele was also significantly associated with increased frequency of CD38-positive CLL. We have also examined IRF4 SNPs in linkage disequilbrium with rs872071 and these remain prognostically informative. IRF4 is expressed in germinal centre and post-germinal centre B-cells committed to plasma cell differentiation, and has a complex role in regulating B-cell maturation and homeostasis. Function of the rs870271 polymorphic variant remains undetermined, although given its location in the 3′UTR we speculate that it may affect gene expression, RNA stability or translation efficiency. The 3′ UTR of IRF4 includes several established and putative miRNA binding sites; at least two of these are bound by miRNAs giving rise to a reduction in IRF4 protein expression in germinal centre B-cells. In summary, this study identifies a common variant in IRF4, previously associated with increased risk of developing CLL, as a predictor of a more aggressive disease course and reduced time to first treatment. The strong association with CD38 expression and the identification of a putative IRF4 binding site in the CD38 gene immediately upstream of the transcriptional start site suggests a plausible mechanism by which IRF4 status could affect prognosis. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4723.

Imatinib has become first-line therapy in chronic myeloid leukaemia (CML), but recent studies suggest that more than one-third of patients fail imatinib over 2–3 years. (de Lavallade et al, 2008; Lucas et al, 2008) It would be clinically helpful to prospectively identify patients unlikely to achieve a complete cytogenetic response (CCRe) following imatinib treatment. Prognostic scoring methods, such as the Sokal score, activity of the imatinib uptake transporter, hOCT1 (human organic cation transporter 1) (Wang et al, 2008) and BCR-ABL1 transcript type (Lucas et al, 2009) have been shown to correlate with clinical outcome. However, none of these parameters are sufficiently powerful to reliably prospectively predict patients destined to fare poorly. The phosphorylation status of CrkL has been identified as a surrogate marker of BCR-ABL1 tyrosine kinase activity (White et al, 2005), because BCR-ABL1 tyrosine kinase activity cannot be reliability detected in CML patient samples (Patel et al, 2007). White et al (2005) showed, by Western blotting, that changes in the level of CrkL phosphorylation after 1 month of imatinib treatment correlated with clinical outcome. Flow cytometry has the advantage that it requires fewer patient cells (∼103 cells) than Western blotting, produces quantitative values, and results can be obtained rapidly. Additionally this methodology could be utilized more easily at other laboratories. A representative FACS histogram of a patient sample is shown in Fig 1. Patient characteristics and optimization of pCrKL and CrKL detection by flow cytometry. (A) Summary of patients characteristics. (B) Representative FACS plot showing pCrKL and CrKL (C) in a newly diagnosed CML patient and changes following in vitro treatment with 5 μmol/l imatinib for 24 h. Our study demonstrated that measuring pCrkL levels alone did not correlate with clinical outcome (Fig 2A). Hundred per cent of patients with a pre-treatment pCrkL/CrkL ratio <25 achieved CCRe following 12 months of imatinib treatment (Fig 2B). Of these, 75% of patients achieved their CCRe within 6 months. No patient whose pCrkL/CrkL ratio was greater than 25 achieved CCRe following 12 months of imatinib treatment (P = 0·0003). pCrKL, CrKL and pCrKL/CrKL ratio level at diagnosis and clinical outcome. FACS assessment of pCrKL (A) and pCrKL/CrKL ratio (B) in untreated newly diagnosed CML patients, stratified by clinical outcome following 12 months of imatinib treatment. (C) pCrKL/CrKL ratio at diagnosis and clinical outcome at 12 months for imatinib- and nilotinib-treated patients. Patients with a pCrKL/CrKL ratio below 25 responded to imatinib treatment. Six out of 14 imatinib-treated patients failed to achieve a CCRe following imatinib treatment. Three switched to the second generation tyrosine kinase inhibitor nilotinib and the other three remain on imatinib. No BCR-ABL1 kinase domain mutations were detected in patients who failed to achieve a CCRe by 12 months. The remaining eight patients, with a diagnostic pCrkL/CrkL ratio below 25 and a maximum follow up of 2 years, remain in CCRe. During this study six patients commenced nilotinib treatment at initial diagnosis. Their pCrkL/CrkL MFI ratios at diagnosis ranged from 7·5 to 47·3 Fig 2C. Following 12 months of treatment all achieved a complete cytogenetic response. Patients with a pCrkL/CrkL MFI ratio greater than 25 who were treated with nilotinib achieved a CCRe compared to 0% of imatinib-treated patients (P = 0·03). This is probably because nilotinib is more potent BCR-ABL1 tyrosine kinase inhibitor (O'Hare et al, 2005). These data suggest that patients with high BCR-ABL1 tyrosine kinase activity (pCrkL/CrkL MFI ratio >25) have a low probability of responding to imatinib, but may nevertheless achieve a complete cytogenetic response on nilotinib treatment. All nilotinib-treated patients remained in CCRe with continued treatment. In addition, serial monitoring of the trends in the pCrkL/CrkL ratio following commencement of imatinib treatment had no predictive value. We analysed the diagnostic pCrkL/CrkL ratio data for both imatinib- and nilotinib-treated patients in relation to other clinical parameters such as white blood cell count at diagnosis, Sokal score and hOCT1 (for imatinib-treated patients only), (Davies et al, 2009) and found no correlation. This suggests that the pCrkL/CrkL ratio is an independent biomarker of clinical outcome for imatinib treated patients. It is interesting to speculate that the response to imatinib therapy is related to BCR-ABL1 tyrosine kinase activity present at diagnosis. A fixed dose of imatinib may therefore only be able to suppress the lower kinase activity i.e. pCrkL/CrkL ratio <25; while nilotinib, which is known to be a more potent BCR-ABL1 kinase inhibitor than imatinib (Kantarjian et al, 2007), may suppress the BCR-ABL1 tyrosine kinase activity to a greater proportional extent. This may explain the higher incidence of CCRe following treatment with nilotinib. In conclusion, the pCrkL/CrkL ratio prior to commencement of treatment is a potent predictor of clinical outcome in patients treated with imatinib. Its role as a predictive biomarker of response to tyrosine kinase inhibitor therapy merits further investigation in larger series, though it is essential that this is performed on freshly isolated cells prior to any treatment. The authors would like to thank the University of Liverpool Biobank staff Joanna Middleton, Melanie Oates and Alison Hayes for their help and support during this project. CML, RJH and REC designed the study and wrote the manuscript. CML and AG performed the laboratory work for this study. KK and SW were responsible for taking patient samples and providing clinical information. REC was the principal investigator. The authors reported no potential conflicts of interest.

To identify new risk variants for chronic lymphocytic leukemia (CLL), we conducted a genome-wide association study of 299,983 tagging SNPs, with validation in four additional series totaling 2,503 cases and 5,789 controls. We identified four new risk loci for CLL at 2q37.3 (rs757978, FARP2; odds ratio (OR) = 1.39; P = 2.11 x 10(-9)), 8q24.21 (rs2456449; OR = 1.26; P = 7.84 x 10(-10)), 15q21.3 (rs7169431; OR = 1.36; P = 4.74 x 10(-7)) and 16q24.1 (rs305061; OR = 1.22; P = 3.60 x 10(-7)). We also found evidence for risk loci at 15q25.2 (rs783540, CPEB1; OR = 1.18; P = 3.67 x 10(-6)) and 18q21.1 (rs1036935; OR = 1.22; P = 2.28 x 10(-6)). These data provide further evidence for genetic susceptibility to this B-cell hematological malignancy.

We conducted a genome-wide association study of 299,983 tagging SNPs for chronic lymphocytic leukemia (CLL) and performed validation in two additional series totaling 1,529 cases and 3,115 controls. We identified six previously unreported CLL risk loci at 2q13 (rs17483466; P = 2.36 x 10(-10)), 2q37.1 (rs13397985, SP140; P = 5.40 x 10(-10)), 6p25.3 (rs872071, IRF4; P = 1.91 x 10(-20)), 11q24.1 (rs735665; P = 3.78 x 10(-12)), 15q23 (rs7176508; P = 4.54 x 10(-12)) and 19q13.32 (rs11083846, PRKD2; P = 3.96 x 10(-9)). These data provide the first evidence for the existence of common, low-penetrance susceptibility to a hematological malignancy and new insights into disease causation in CLL.

AbstractChemokine-induced activation of α4β1 and αLβ2 integrins (by conformational change and clustering) is required for lymphocyte transendothelial migration (TEM) and entry into lymph nodes. We have previously reported that chemokine-induced TEM is defective in chronic lymphocytic leukemia (CLL) and that this defect is a result of failure of the chemokine to induce polar clustering of αLβ2; engagement of α4β1 and autocrine vascular endothelial growth factor (VEGF) restore clustering and TEM. The aim of the present study was to characterize the nature of this defect in αLβ2 activation and determine how it is corrected. We show here that the αLβ2 of CLL cells is already in variably activated conformations, which are not further altered by chemokine treatment. Importantly, such treatment usually does not cause an increase in the GTP-loading of Rap1, a GTPase central to chemokine-induced activation of integrins. Furthermore, we show that this defect in Rap1 GTP-loading is at the level of the GTPase and is corrected in CLL cells cultured in the absence of exogenous stimuli, suggesting that the defect is the result of in vivo stimulation. Finally, we show that, because Rap1-induced activation of both α4β1 and αLβ2 is defective, autocrine VEGF and chemokine are necessary to activate α4β1 for ligand binding. Subsequently, this binding and both VEGF and chemokine stimulation are all needed for αLβ2 activation for motility and TEM. The present study not only clarifies the nature of the αLβ2 defect of CLL cells but is the first to implicate activation of Rap1 in the pathophysiology of CLL. [Cancer Res 2008;68(20):8429–36]

We have previously shown that imatinib uptake into chronic myeloid leukemia (CML) cells is dependent on human organic cation transporter 1 (hOCT1; SLC22A1), and that low hOCT1 expression is an important determinant of clinical outcome to imatinib treatment. We hypothesized that dasatinib might be transported differently than imatinib, possibly accounting for its favorable effects in imatinib-resistant patients. (14)C-dasatinib uptake was greater in KCL22-transfected cells with pcDNA3-hOCT1 plasmid (high hOCT1-expressing cells) than in control cells (P = .02). However, hOCT inhibitors did not decrease dasatinib uptake into either control or primary cells, in contrast to their block on imatinib uptake. Dasatinib decreased the level of phosphorylated CrkL to 49.9% in control and 40.3% in high hOCT1-expressing cells. Dasatinib efflux was investigated in confluent ABCB1-transfected MDCKII cell monolayers. Both dasatinib and imatinib were transported from the basal to the apical layer, indicating that they were transported by ABCB1, which was confirmed using the ABCB1 inhibitor PSC833 (P = .001 and P < .001, respectively). Compared with imatinib, dasatinib achieved superior intracellular levels and BCR-ABL suppression even in cells with low or blocked hOCT1. Efflux of dasatinib and imatinib appear similar via ABCB1. Dasatinib may therefore offer an advantage over imatinib in patients with low hOCT1 expression.

Tyrosine kinase inhibitors (TKIs) that preferentially inhibit the oncogenic Bcr-Abl fusion protein have radically changed the treatment of chronic myeloid leukaemia (CML). Imatinib mesylate ((IM) Gleevec®, Glivec®, STI571; Novartis) has been shown to induce almost complete hematological responses in patients after 12 months and major cytogenetic responses in 69%. However, the proportion of patients achieving molecular responses or having undetectable Bcr-Abl transcripts remains low. In addition to this failure to induce molecular response, resistance occurs in 10–15% of patients. Nilotinib ((NIL) AMN107; Novartis) has approximately 20-fold greater potency in in vitro assays but despite this improved potency in vitro and on bulk primary CML cells we have found that NIL is no better than IM in inducing apoptosis of the primitive CD34+ CML cell population. We previously showed that as with IM, CD34+ CML cells persist after NIL treatment and that NIL also has an anti-proliferative rather than pro-apoptotic effect, resulting in the accumulation of quiescent CD34+ cells. It has been proposed that this population of TKI-insensitive primitive cells may form a pool of disease in patients under treatment and contribute to the Bcr-Abl molecular signal detected in the majority of patients. This population must therefore, be specifically targeted in order to eradicate the disease in patients and to result in cure rather than control of the disease. One possible reason for the failure of NIL to kill CD34+ CML cells is that the cells do not accumulate sufficient intracellular levels of the drug due to either inadequate active uptake via the SLC transporter family, or to efflux via multidrug resistance proteins of the ATP-binding cassette (ABC) family. In order to determine the interaction of NIL with major clinically implicated drug transporters we have investigated interactions with ABCB1, MRP1 (ABCC1), ABCG2 and hOCT1 in CML cell lines and primitive (CD34+) primary CML cells.

4314 The tumour suppressor protein p53 and the DNA damage activated kinase, ataxia telangiectasia mutated (ATM), play crucial roles in the DNA damage response pathway. B-cell Chronic Lymphocytic leukaemia (CLL) patients with p53 or ATM gene deletions (del(17p) and del(11q), respectively) and/or mutations are chemoresistant and have shorter survival. DNA-dependent protein kinase (DNA-PK) is also a DNA damage activated kinase essential for the repair of DNA double strand breaks. Here, we have investigated the relationships between p53 functionality, p53 gene deletion and mutational status, DNA-PK catalytic subunit (DNA-PKcs) expression and chemo-resistance in a cohort of well-characterised CLL cases. We used WAVE analysis (n = 72) followed by direct sequencing to screen for mutations in exons 5 through 8 of p53. In selected cases, p53 function was assessed by ionising radiation-induced up-regulation of p21, and prolonged half-life of p53 (associated with p53 mutation) using flow cytometric analysis. Resistance to fludarabine and chlorambucil was assessed (n = 54) in ex vivo cytotoxicity assays using patient-derived CLL cells, together with the ability of a specific and potent inhibitor of DNA-PK (NU7441) to sensitise CLL cells these agents.
 Six cases harboured del(17p), and sequencing studies showed that all these cases displayed mutations on the remaining allele. Mutations occurred in exons 5 - 8, which comprise the DNA-binding domain and may ablate the ability of p53 to transcriptionally activate target genes. p53 dysfunction correlated with the presence of p53 mutations.
 Western blotting analyses confirmed that DNA-PKcs levels were consistently higher in the cases that harboured del(17p), compared to those with del (13q) (p = 0.01). Cytotoxicity assays showed that del(17p) combined with the presence of p53 mutation increased resistance to chlorambucil and fludarabine, compared to del(13q) cases (XTT assay). NU7441 sensitized CLL cells to fludarabine (2-10 fold) and chlorambucil (2-20 fold) respectively (in some cases, no sensitisation was observed). Kaplan Meier analysis confirmed the expected association between short time to treatment (TTT) and presence of del(17p), with a median TTT of 17 months versus 72 months for cases without del (17p). These data demonstrate a strong correlation between the presence of del(17p), p53 mutation and increased levels of DNA-PKcs. p53 mutation also occurred in the absence of del(17p) (n = 16), and although this did not necessarily confer a poor prognosis, p53 mutation per se did correlate with high levels of DNA-PKcs. We speculate that increased DNA-PK activity may contribute to genomic instability in CLL, and hence promote disease progression. Targeted inhibition of DNA-PK may prove a valuable addition to CLL therapeutic regimes.

In chronic myeloid leukaemia (CML), resistance to imatinib is an important clinical issue. We have previously shown that imatinib uptake into CML cells and cell lines is dependent on the uptake transporter human Organic Cation Transporter 1 (hOCT1; SLCA22). Furthermore, in clinical samples, low hOCT1 expression is an important mechanism of imatinib resistance (Wang L et al, Clinical Pharmacology and Therapeutics; epub June 13th 2007). Dasatinib is a second generation novel tyrosine kinase inhibitor that is effective in many imatinib-resistant patients. Its mechanism of transport into and out of CML cells has not been previously investigated. We hypothesised that dasatinib might be transported differently to imatinib, which might account for its favourable effects in imatinib-resistant patients. The CML cell line KCL22 was selected for transfection work as it has a particularly low basal level of hOCT1 expression. KCL22 cells were transfected with pcDNA3-hOCT1 plasmid (kind gift of D Gründemann, Germany) and stable lines were selected with high hOCT1 expression. The uptake of radiolabelled dasatinib (kind gift from Bristol Myers Squibb) was greater in high-hOCT1 expressing cells than in mock transfected cells (p=0.0149, n=3). However, prazosin and amantadine, both inhibitors of hOCT transport, did not decrease dasatinib uptake into mock transfected KCL22 cells, in sharp contrast to the block on imatinib uptake seen with both these inhibitors. Dasatinib decreased the level of phosphorylated CRKL (surrogate marker for BCR-ABL) by 49.9% in mock transfected KCL22 cells and 40.3% in high-hOCT1 expressing cells. The distribution coefficient (logD) of dasatinib between 1-octanol and HEPES medium was 2.05, compared with 0.81 for imatinib, demonstrating that dasatinib is more lipophilic. The efflux of dasatinib was investigated in confluent monolayers of Madin-Darby canine kidney (MDCKII) cells on a semipermeable membrane. These cells stably express ABCB1 (MDR1) on their apical but not their basal aspect. Both dasatinib and imatinib were transported from the basal to the apical layer, indicating ABCB1 transporter-mediated efflux of both drugs (p=0.001, p<0.0001, respectively). Addition of the ABCB1 inhibitor PSC833 blocked transport of both drugs (p=0.0013, p<0.0001 respectively). Taken together, the data are consistent with the view that dasatinib, unlike imatinib, may achieve adequate intracellular levels and BCR-ABL suppression even in cells with low or blocked hOCT1 function. However, in cells with high hOCT1 expression, dasatinib uptake may be augmented. Efflux of dasatinib and imatinib appear similar and via ABCB1. Dasatinib may be useful therapeutically in patients with imatinib resistance related to low hOCT1 expression.

We have previously identified the presence of Ras/Raf-independent constitutive activation of extracellular signal-regulated kinase (ERK) in the hairy cells (HCs) of hairy cell leukemia. The aim of the present study was to characterize the signaling components involved in this activation and their relationship to the reported activation of Rac1. We found that both Rac1 and ERK activation in HCs are downstream of active Src and protein kinase C (PKC). Inhibition with toxin B showed that Rac1 plays no role in ERK activation in HCs. However, toxin B inhibited p60src and the Rac1-GEF Vav, demonstrating a positive feedback/activation of p60src by Rac1. Treatment with specific small interfering RNA for various PKC isoforms, or with PKC isoform-specific inhibitors, demonstrated a central role for PKCepsilon in the constitutive activation of Rac1 and ERK in HCs. PKCepsilon and active ERK were mutually associated and co-localized with mitochondria in HCs. Furthermore, active PKCepsilon was nitrated on tyrosine, pointing to a reactive oxygen species-dependent mechanism of activation. By being involved in activation of ERK and Rac1, PKCepsilon plays roles in both the survival of HCs and in the cytoskeletal dynamics responsible for the distinctive morphology and tissue homing of these cells. Our study therefore describes novel aspects of signaling important for the pathogenesis of hairy cell leukemia.