Establishing the posh scaffold as a novel therapeutic target for treatment of B cell leukemia

2020 ◽  
Author(s):  
◽  
Leah Nicole Cardwell
Keyword(s):  
B Cells ◽  
B Cell ◽  
Cell Survival ◽  
Therapeutic Target ◽  
Mitogen Activated Protein Kinase ◽  
Rapid Progression ◽  
Cell Leukemia ◽  
Jnk Signaling ◽  
B Cell Leukemia ◽  
Novel Therapeutic

[ACCESS RESTRICTED TO THE UNIVERSITY OF MISSOURI-COLUMBIA AT REQUEST OF AUTHOR.] More than 60,000 new cases of leukemia will be diagnosed in the U.S. this year. B cells comprise 10 [percent] of acute lymphoblastic leukemias (B-ALL) and almost all chronic lymphocytic leukemias (B-CLL). B-ALL are typified by rapid progression (weeks) and are predominant in children. B-CLL is characterized by slow progression (months) and are predominant in adults. Despite advances in current treatment therapies, over 23,000 people will die from leukemia this year. Thus, the focus of this research was to establish a novel therapeutic target for treatment of B cell leukemia. In B cells, intracellular c-Jun N-terminal kinase (JNK) signaling, a member of the mitogen-activated protein kinase (MAPK) family, regulates cell survival and death in response to cellular stimuli. Cell stressors predominantly induce sustained JNK activity and apoptosis, with chemotherapeutic drugs initiating JNK-mediated apoptosis in leukemic B cells. However, stimuli specific modulations in JNK activity also results in cell survival, proliferation, and differentiation, with several B-ALL/B-CLL exhibiting overactive JNK signaling for survival. JNK signaling is activated via a three-tiered cascade composed of MAP3K-MAP2K-MAPK (JNK) proteins. Scaffold-mediated formation of unique multi-protein-containing complexes regulate cell-type and stimuli-specific signaling cascades. Plenty of SH3 domains (POSH) is a scaffold protein that acts as a platform for concentrated assembly of unique MAPK-containing complexes to efficiently mediate pro- or anti-apoptotic JNK signaling outcomes. We are the first to characterize the function of the POSH scaffold in both healthy and leukemic B cells. Herein, we report that disruption of POSH scaffold function induces JNK-mediated apoptosis of several diverse acute and chronic B cell leukemias, healthy splenic B cells, but with limited survival functions in healthy bone marrow B cells, via use of our unique competitive inhibitor termed Tat-POSH-inhibitor. In addition to these findings, we also report experimental insights into POSH mechanistic functions, generation of POSH knockout mice, and the generation and characterization of novel cell-targeting aptamer- and micelle-based methods of Tat-POSH-inhibitor delivery. The work presented in this dissertation aims to establish POSH as a novel therapeutic target for treatment of acute and chronic B cell leukemias.

Expression of CD13/aminopeptidase N in precursor B-cell leukemia: role in growth regulation of B cells

2009 ◽  
Vol 59 (1) ◽  
pp. 125-135 ◽  
Author(s):  
Ankit Saxena ◽  
Ambak Rai ◽  
Vinod Raina ◽  
Tulika Seth ◽  
Dipendra Kumar Mitra
Keyword(s):  
B Cells ◽  
B Cell ◽  
Growth Regulation ◽  
Aminopeptidase N ◽  
Cell Leukemia ◽  
B Cell Leukemia

Frequent Loss of the SLP-65 Adapter Protein in Childhood Acute B-Precursor Lymphoblastic Leukemia (ALL) with TEL/AML1 Rearrangement.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 741-741 ◽  
Author(s):  
Arndt Borkhardt ◽  
Christine Damm-Welk ◽  
Thomas Wossning ◽  
Bettina Storch ◽  
Uta Fuchs ◽  
...  
Keyword(s):  
Cell Differentiation ◽  
B Cells ◽  
Tumor Suppressor ◽  
Protein Expression ◽  
B Cell ◽  
Cell Lines ◽  
Sh2 Domain ◽  
B Cell Differentiation ◽  
Cell Leukemia ◽  
B Cell Leukemia

Abstract The adaptor protein SLP-65 plays an essential role during B cell differentiation. A crucial consequence of SLP-65 deficiency in mice is a high incidence of pre-B-cell leukemia, suggesting a tumor suppressor role for SLP-65 in pre-B-cells. While the link between SLP-65 deficiency and leukemia development is established in mice, experiments mainly using microarrays for gene expression profiling suggested normal expression of SLP-65 in human precursor B-cell ALL. This analysis however does not discriminate between normal and aberrant SLP-65 transcripts with the latter being unable to generate functional protein. To examine the correlation between SLP-65 deficiency and childhood precursor B-cell ALL, we determined SLP-65 expression in 119 precursor B-cell ALL samples by both RNA and protein methods. The expression of SLP-65 was compared to clinical and laboratory findings, cytogenetics as well as to the outcome data within this uniformly treated cohort of patients. Loss of slp-65 protein was significantly associated with the occurrence of the TEL/AML1 rearrangement (p=0.026) but not with any other clinical or cytogenetic feature. We found a profound disconnection between slp-65 mRNA and protein expression in 38 out of the 119 leukemic samples pointing to a posttranscriptional regulation of slp-65 (Table). To confirm that SLP-65 transcript expression does not automatically correlate with its protein expression, we analyzed a panel of human cell lines derived from precursor B-cell ALL patients. The cell lines HPB-NULL and BV-173 showed a deficiency in SLP-65 protein expression, although SLP-65 transcripts can easily be detected in both lines. Together, the data suggest that SLP-65 expression might be regulated at the posttranscriptional level and that the presence of SLP-65 transcripts does not necessarily lead to SLP-65 protein and function. In one particular patient, we found a truncated slp-65 transcript and the predicted slp-65 protein lacks its SH2 domain. We tested whether this SLP-65 protein lacking the SH2 domain is functional in pre-B cells. To this end, we transfected murine SLP-65 −/− pre-B cells with retroviral constructs for either wild-type (wt SLP-65) or truncated SLP-65 (SLP-65delSH2) and analysed pre-BCR downregulation, Ca2+ release and pre-B cell differentiation. The results showed that, in contrast to wt SLP-65, SLP-65delSH2 failed to induce any effects in the performed experiments. Together with previous findings showing that SLP-65-deficient mice develop pre-B cell leukemia, the data suggest that SLP-65 acts as a tumor suppressor that limits pre-B cell proliferation by inducing differentiation. Disconnection between slp-65 transcripts and protein expression total slp-65 protein+ (51 patients) slp-65 protein weak (19 patients) slp-65 protein- (49 patients) PCR+ 108 51(9 TEL/AML+, 42 TEL/AML-) 19 (9 TEL/AML+, 10 TEL/AML-) 38 (15 TEL/AML+, 23 TEL/AML-) PCR- 11 0 0 11 (T-ALL)

scholarly journals Nurselike cells express BAFF and APRIL, which can promote survival of chronic lymphocytic leukemia cells via a paracrine pathway distinct from that of SDF-1α

Blood ◽  
2005 ◽  
Vol 106 (3) ◽  
pp. 1012-1020 ◽  
Author(s):  
Mitsufumi Nishio ◽  
Tomoyuki Endo ◽  
Nobuhiro Tsukada ◽  
Junko Ohata ◽  
Shinichi Kitada ◽  
...  
Keyword(s):  
B Cells ◽  
Chronic Lymphocytic Leukemia ◽  
B Cell ◽  
Cell Survival ◽  
Cell Activation ◽  
Leukemia Cell ◽  
Lymphocytic Leukemia ◽  
Mitogen Activated Protein Kinase ◽  
Mitogen Activated Protein ◽  
Stromal Cell Derived Factor

AbstractWe examined expression of B cell–activating factor of the tumor necrosis factor (TNF) family (BAFF) and a proliferation-inducing ligand (APRIL) on chronic lymphocytic leukemia (CLL) B cells and nurselike cells (NLCs), which differentiate from CD14+ cells when cultured with CLL B cells. NLCs expressed significantly higher levels of APRIL than monocytes and significantly higher levels of BAFF and APRIL than CLL B cells. Also, the viability of CLL B cells cultured with NLCs was significantly reduced when CLL B cells were cultured with decoy receptor of B-cell maturation antigen (BCMA), which can bind both BAFF and APRIL, but not with BAFF receptor:Fc (BAFF-R:Fc), which binds only to BAFF. The effect(s) of BAFF or APRIL on leukemia cell survival appeared additive and distinct from that of stromal cell–derived factor-1α (SDF-1α), which in contrast to BAFF or APRIL induced leukemia cell phosphorylation of p44/42 mitogen-activated protein kinase (extracellular signal-regulated kinase-1/2 [ERK1/2]) and AKT. Conversely, BAFF and APRIL, but not SDF-1α, induced CLL-cell activation of the nuclear factor–κB1 (NF-κB1) and enhanced CLL-cell expression of the antiapoptotic protein Mcl-1. However, BAFF, but not APRIL, also induced CLL-cell activation of NF-κB2. We conclude that BAFF and APRIL from NLCs can function in a paracrine manner to support leukemia cell survival via mechanisms that are distinct from those of SDF-1α, indicating that NLCs use multiple distinct pathways to support CLL-cell survival.

scholarly journals BAFF regulates B cell survival by downregulating the BH3-only family member Bim via the ERK pathway

2005 ◽  
Vol 202 (10) ◽  
pp. 1363-1374 ◽  
Author(s):  
Andrew Craxton ◽  
Kevin E. Draves ◽  
Adriana Gruppi ◽  
Edward A. Clark
Keyword(s):  
B Cells ◽  
B Cell ◽  
Cell Survival ◽  
Mitogen Activated Protein Kinase ◽  
Marginal Zone B Cells ◽  
B Cell Survival ◽  
Extracellular Signal ◽  
Mitogen Activated Protein ◽  
B Cell Homeostasis ◽  
Induced Apoptosis

The B cell activating factor belonging to the tumor necrosis factor family (BAFF) is required for B cell survival and maturation. The mechanisms by which BAFF mediates B cell survival are less understood. We found that BAFF and a proliferation-inducing ligand (APRIL), which are related, block B cell antigen receptor (BCR)–induced apoptosis upstream of mitochondrial damage, which is consistent with a role for Bcl-2 family proteins. BCR ligation strongly increased expression of the proapoptotic Bcl-2 homology 3–only Bcl-2 protein Bim in both WEHI-231 and splenic B cells, and increases in Bim were reversed by BAFF or APRIL. Small interfering RNA vector–mediated suppression of Bim blocked BCR-induced apoptosis. BAFF also induced Bim phosphorylation and inhibited BCR-induced association of Bim with Bcl-2. BAFF induced delayed but sustained stimulation of extracellular signal–regulated kinase (ERK) and its activators, mitogen-activated protein kinase/ERK activating kinase (MEK) and c-Raf, and MEK inhibitors promoted accumulation and dephosphorylation of Bim. These results suggest that BAFF inhibits BCR-induced death by down-regulating Bim via sustained ERK activation, demonstrating that BAFF directly regulates Bim function. Although transitional immature type 1 (T1) B cell numbers are normal in Bim−/− mice, T2 and follicular mature B cells are elevated and marginal zone B cells are reduced. Our results suggest that mature B cell homeostasis is maintained by BAFF-mediated regulation of Bim.

ROR1 Expression Accelerates Leukemia Development in RORxTCL1 Transgenic Mice,

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 3905-3905
Author(s):  
George F. Widhopf ◽  
Bing Cui ◽  
Esther Avery ◽  
George Chen ◽  
Masato Obara ◽  
...  
Keyword(s):  
B Cells ◽  
B Cell ◽  
Adoptive Transfer ◽  
Conflicts Of Interest ◽  
Leukemia Cell ◽  
Leukemia Cells ◽  
Terminal Deoxynucleotidyl Transferase ◽  
Cell Turnover ◽  
Cell Leukemia ◽  
B Cell Leukemia

Abstract Abstract 3905 ROR1 is a receptor tyrosine kinase-like orphan receptor and an oncofetal protein that is expressed on chronic lymphocytic leukemia (CLL) B cells, but not on normal B cells or most other adult tissues. The leukemia-associated expression of ROR1 suggests that it potentially could contribute to the development and/or progression of CLL. To investigate its functional significance in the development and/or progression of CLL, we generated B6 mice transgenic (Tg) for human ROR1 (hROR1) under the control of the murine Ig promoter/enhancer, which drives B-cell-restricted expression of hROR1. These mice developed increased numbers of splenic B lymphocytes relative to that of control littermates lacking the hROR1 transgene; a few of these animals also developed hROR1+/CD5+/B220low B cell leukemia resembling human CLL at 15–18 months of age. We crossed hROR1-Tg mice with B6 Eμ-TCL1-Tg mice (TCL1), which at 7 months of age develop CD5+B220low leukemia B cells detectable in the blood and resembling human CLL cells except lacking expression of mouse ROR1. We found the F1 animals with both transgenes (ROR1XTCL1) developed hROR1+/CD5+/B220low B-cell CLL at a significantly younger median age than did littermate-control mice having either transgene alone. Comparison of the number of CD5+B220low leukemia B cells in ROR1xTCL1 or TCL1 Tg mice at five, six, and seven months of age demonstrated that ROR1xTCL1 Tg mice have significantly higher median percentages of circulating leukemic cells in the blood compared to that of age-matched TCL1 Tg mice (Welchs's t test based on the average of all 3 measurements, p=0.038). ROR1xTCL1 leukemia B cells also expressed higher levels of hROR1 than non-leukemia B cells of ROR1 Tg mice. To confirm that ROR1 enhances leukemia-cell expansion, we adoptively transferred CD5+B220low leukemia B cells from ROR1xTCL1 Tg mice into syngeneic ROR1-Tg mice. Adoptive transfer of equal numbers (1×105) of leukemia B cells from ROR1xTCL1 or TCL1 Tg mice resulted in transfer of leukemia in both cases without requiring prior conditioning of recipient animals. However, animals engrafted with ROR1xTCL1 leukemia cells developed more aggressive disease and more marked splenomegaly than did animals engrafted with TCL1 leukemia cells. Moreover, eight weeks after adoptive transfer, animals engrafted with ROR1xTCL1 leukemia cells had ≥4-fold greater median numbers of neoplastic B cells in the spleen (e.g. 4 × 108) than did animals engrafted with TCL1 leukemia cells. In vivo labeling studies with bromodeoxyuridine (BrdU), measurement of the relative proportions of Ki-67-positive leukemia cells from either type of adoptive host animals, and use of the terminal deoxynucleotidyl transferase (dUTP) nick end-labeling (TUNEL) assay to detect cells that had undergone apoptosis. These studies revealed that animals engrafted with hROR1+ leukemia cells had significantly lower-rates of leukemia-cell turnover due to higher rates of cell proliferation and lower rates of apoptosis than that animals engrafted with TCL1 leukemia cells. These studies indicate that hROR1 can accelerate development of de novo B-cell leukemia, lower the rate of leukemia-cell turnover, and enhance disease progression in this model system. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Dynamic Assembly of a Feedback Complex to Regulate Oncogenic B-Cell Receptor-Signaling

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 393-393
Author(s):  
Jaewoong Lee ◽  
Kohei Kume ◽  
Zhengshan Chen ◽  
Gang Xiao ◽  
Kadriye Nehir Nehir Cosgun ◽  
...  
Keyword(s):  
B Cells ◽  
B Cell ◽  
Research Funding ◽  
Cytoplasmic Tail ◽  
Cell Receptor ◽  
Transplant Recipients ◽  
Cell Leukemia ◽  
B Cell Malignancies ◽  
Bcr Signaling ◽  
B Cell Leukemia

Background: Studying gene expression and clinical outcome data from 136 clinical trials for patients with cancer (~21,000 patients with 26 cancer types), we found CD25 as one of the strongest predictors of poor clinical outcome in patients with B-cell malignancies, but not in other cancer types. This was unexpected because CD25 is known as one of three chains of the IL2 receptor on T-cells and NK-cells. Interleukin-2 (IL2) functions as essential T-cell growth factor. IL2 signals through b- and g-, but not a-chains (CD25) of its heterotrimeric receptor. CD25-deficiency causes lymphoproliferation and autoimmunity, however, its mechanistic role is unclear. Results: Our experiments based on genetic mouse models and engineered patient-derived B-cell leukemia and lymphoma xenografts revealed that CD25 expressed on B-cells is not an IL2 receptor chain, but in fact binds downstream signaling molecules of the B-cell receptor (BCR). Through these interactions, CD25 mediates negative feedback to BCR signaling in response to antigen-encounter in normal B-cells. Defects in CD25-/-B-cells were not replicated in mice that express CD25 but lack expression of the IL2 cytokine. These findings demonstrate IL2-independent functions of CD25 in B-cells and B-cell derived leukemia and lymphoma. To comprehensively study the interactome of the short cytoplasmic tail of CD25, we performed proximity-dependent biotin identification (BioID). This analysis revealed that the CD25 tail exerts negative feedback control through recruitment of the PKCβ-scaffold RACK1 and the inhibitory phosphatase SHIP1 (see schematic, left). Interestingly, the cytoplasmic tail of CD25 harbors a PKCβ-substrate motif and mutation of a central serine residue (S268) to A268 compromised interactions with PKCβ, its scaffold RACK1 and SHIP1, demonstrating that feedback control was dependent on PKCβ-mediated phosphorylation of CD25-S268. A genetic observation in a family with monogenic autoimmunity confirmed the functional importance of the cytoplasmic CD25-tail motif: a mutation immediately preceding S268 compromised CD25-surface translocation, which was restored by homology-directed repair of the S268. In vitro kinase assay with 62 candidate kinases against recombinant cytoplasmic tail of CD25-S268 or -A268 identified PKCβ as top-ranking kinase hit for CD25-S268 but not CD25-A268. Our genetic studies revealed that PKCβ is required for cell-membrane translocation of CD25, but also transcriptional expression of CD25 via NF-κB activation. Therefore, PKCβ act as critical effector molecule downstream of CD25 to mediate B-cell selection during normal B-cell development and calibrate oncogenic BCR signaling in B-cell tumors. In B-cell malignancies, BCR-dependent survival and proliferation signals are often substituted by oncogenic BCR-mimics (e.g. BCR-ABL1, JAK2, BRAFV600E, LMP2A, CD79B mutations; see schematic, right). Accordingly, we identified CD25 surface-expression as biomarker of oncogenic BCR-signaling and predictor of poor clinical outcomes. CD25-/-B-cell leukemia failed to initiate fatal disease in transplant recipients. Owing to imbalances of oncogenic BCR-signaling and p53-checkpoint activation, CD25-/- B-cell leukemia failed to initiate fatal disease in transplant recipients. In patient-derived xenograft models of drug-resistant B-cell malignancies, treatment with a CD25-specific antibody drug-conjugate (ADCT-301) extended survival of transplant recipients or eradicated disease. These findings identified CD25 as previously unrecognized feedback regulator of oncogenic BCR-signaling and provide a rationale for therapeutic targeting of CD25 in refractory B-cell malignancies. Figure Disclosures Zammarchi: ADC Therapeutics: Employment. Van Berkel:ADC Therapeutics: Research Funding. Melnick:Constellation: Consultancy; Janssen: Research Funding; Epizyme: Consultancy. Luger:Celgene: Research Funding; Cyslacel: Research Funding; Pfizer: Honoraria; Seattle Genetics: Research Funding; Agios: Honoraria; Ariad: Research Funding; Biosight: Research Funding; Kura: Research Funding; Onconova: Research Funding; Genetech: Research Funding; Jazz: Honoraria; Daichi Sankyo: Honoraria. Meffre:AbbVie: Consultancy, Other: Grant. Weinstock:Celgene: Research Funding.

scholarly journals A case of human B cell leukemia that implicates an autocrine mechanism in the abnormal growth of Leu 1 B cells.

1986 ◽  
Vol 78 (5) ◽  
pp. 1331-1338 ◽  
Author(s):  
N Kawamura ◽  
A Muraguchi ◽  
A Hori ◽  
Y Horii ◽  
S Mutsuura ◽  
...  
Keyword(s):  
B Cells ◽  
B Cell ◽  
Cell Leukemia ◽  
Abnormal Growth ◽  
B Cell Leukemia ◽  
Human B Cell ◽  
Autocrine Mechanism

Two-Track Epigenetic Remodeling and Backtracking to Embryonic Stem Cell Bivalency in B-Cell Acute Lymphoblastic Leukemias

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 3557-3557
Author(s):  
Seung-Tae Lee ◽  
Marcus O. Muench ◽  
Marina Fomin ◽  
Jianqiao Xiao ◽  
Mi Zhou ◽  
...  
Keyword(s):  
Dna Methylation ◽  
B Cells ◽  
B Cell ◽  
De Novo ◽  
Embryonic Stem ◽  
Cell Leukemia ◽  
Childhood All ◽  
B Cell Leukemia ◽  
Functional Consequences ◽  
De Novo Methylation

Abstract Purpose: We sought to define the drivers and functional consequences of DNA methylation changes in childhood pre-B cell leukemia. A comprehensive analysis of such DNA methylation changes will reveal aspects of the etiology and pathogenesis of leukemias, and suggest potential therapeutic modalities. Background: The epigenome is extensively altered in carcinogenesis, but the proximate causes, functional consequences, and overall patterning of DNA methylation changes in the pediatric leukemias is not well defined. Methods: We bisulfite sequenced at single base pair resolution two common pre-B cell leukemia case DNAs and a pre-B cell control (CD19+/CD34+ pre-B cells from normal marrow), and analyzed DNA methylation by high definition microarray in another 227 subjects. RNA expression was available for 82 samples, including the sequenced pre-B cells and leukemias. Results: Epigenetic alteration of B-ALLs occurred in two tracks: de novo methylation of small functional compartments and demethylation of large inter-compartmental backbones. The de novo methylation occurred preferentially at polycomb targets and binding sites for the transcriptional co-repressor CtBP2. DNA methylation of ETV6-AML1 leukemia was overall equivalent to the control, with high hyperdiploid methylation levels being 4.4% lower. Hierarchical clustering revealed four groups, with ETV6-AML1 and hyperdiploid leukemias enriched within two separate groups. DNA methylation deviations were exaggerated in lamina-associated domains, with differences corresponding to these methylation clusters and/or cytogenetic groups. DNA methylation changes were correlated with gene expression changes, and a key polycomb expression signature was reversed when DNA methylation marks were pharmacologically removed in leukemia cell lines. Discussion: While leukemia cells retain the majority of their developmentally determined DNA methylation patterns, key modifications with leukemogenesis revealed both broad patterns and functional targets with some dependence on known characteristics of pre-B cell leukemias. Our data suggested a pivotal role of polycomb and CTBP2 in de novo methylation, which may be traced back to bivalency status of embryonic stem cells. Driven by these potent epigenetic modulations, suppression of polycomb target genes was observed along with disruption of developmental fate and cell cycle and mismatch repair pathways and altered activities of key upstream regulators. An appreciation and validation of the observed changes will lead to new insights into etiology, pathogenesis, and treatments of childhood ALL. Disclosures No relevant conflicts of interest to declare.

BRAFV600E Induces ABCB1/P-Glycoprotein Expression and Drug Resistance in B-Cells Via AP-1 Activation

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 2477-2477
Author(s):  
Yo-Ting Tsai ◽  
Gerard Lozanski ◽  
Amy M. Lehman ◽  
Ellen Sass ◽  
Erin K. Hertlein ◽  
...  
Keyword(s):  
Drug Resistance ◽  
B Cells ◽  
B Cell ◽  
Cell Line ◽  
Hek293 Cells ◽  
Luciferase Reporter ◽  
Cell Leukemia ◽  
P Glycoprotein ◽  
B Cell Leukemia

Abstract A subset of patients with chronic lymphocytic leukemia (CLL) and nearly all patients with classic hairy cell leukemia (HCL) harbor somatic BRAF activating mutations. However, the pathological role of activated BRAF in B-cell leukemia development and progression remains unclear. In addition, although HCL patients respond well to the BRAFV600E inhibitor vemurafenib, relapses are being observed, suggesting the development of drug resistance in patients with this mutation. Cell line models to study the mechanism of BRAFV600E in B cell leukemia do not exist. Therefore, we utilized the CLL-like B-cell line OSUCLL (PLoS One 2013, 8(10):e76607) to generate cells with doxycycline (dox)-inducible BRAFV600E expression to examine transcriptional and biochemical features induced by this mutation in B-cells. We observed that BRAFV600E induction enhanced proliferation and activation of MAPK signaling in these cells. Microarray using Affymetrix U133 Plus 2.0 GeneChips demonstrated that 235 genes were up-regulated at least 2-fold, and 227 genes were down-regulated at least 2-fold. Several were confirmed by real-time RT-PCR analysis, including ABCB1 (p<0.001, vehicle versus dox-treated). Increased expression of the ABCB1 gene product, P-glycoprotein (P-gp), was also confirmed by immunoblot. This increase resulted in enhanced P-gp function as determined by rhodamine exclusion assays (p<0.005), an effect that was significantly reversed by the P-gp inhibitor verapamil (p<0.005). As an additional demonstration of P-gp function, BRAFV600E induction via dox treatment resulted in a significant increase in resistance to the P-gp substrate vincristine, and the addition of verapamil significantly reduced vincristine resistance. Importantly, pharmacological inhibition of BRAFV600E and MEK, by vemurafenib and CI-1040 respectively, diminished BRAFV600E-induced MAPK pathway activation and enhancement of ABCB1/P-gp expression. To further understand the transcriptional mechanism of BRAFV600E-induced P-gp expression, we performed luciferase assays using a vector containing 1 kb of ABCB1 promoter driving a luciferase reporter, co-transfected into HEK293 cells with either an empty vector or a vector containing BRAFV600E. These assays showed that BRAFV600E expression enhanced luciferase activity (p<0.001), and this effect was down-regulated by vemurafenib and CI-1040 (p<0.001). The role of AP-1 has been reported in ABCB1 regulation. Thus, we conducted electrophoretic mobility shift assays in the BRAFV600E-transfected OSUCLL B-cells, with or without dox treatment, to identify AP-1 factor(s) involved in ABCB1 regulation by BRAFV600E. In these experiments, a supershift in the AP-1 complex was produced by an antibody to JunD in the presence of dox treatment, demonstrating that at least JunD activity is important in this mechanism. Based on these observations, we conclude that BRAFV600E activates AP-1 proteins including JunD to induce ABCB1/P-gp expression and drug resistance in B-cells. This study uncovers a new pathological role for BRAFV600E in B-cell leukemia, and provides further evidence that combination strategies with inhibitors of BRAFV600E and MEK may be beneficial in delaying disease progression and occurrence of resistance to drugs that are substrates of P-gp. Disclosures Byrd: Acerta Pharma BV: Research Funding.

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