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The precise role of mechanistic target of rapamycin complex 1 (mTORC1) during chronic lymphocytic leukemia (CLL) pathogenesis remains to be elucidated. Targeted deletion of mTORC1 component Raptor in adult mice reveals that mTORC1 function is essential for initiation and maintenance of CLL. Raptor-deficient bone marrow-derived PKCα-KR transduced haemopoietic progenitors failed to generate a CLL-like disease in vitro, due to an inability to overcome the mTORC1-mediated block in B cell lineage commitment. Induction of Raptor-deficiency in NSG mice transplanted with Mx1-Raptor BM-derived PKCα-KR transduced cells after disease was established, revealed a reduced CLL-like disease load and a significant increase in survival in the mice. Interestingly in mice transplanted with an aggressive CLL-like disease, rapamycin treatment reduced disease burden more effectively than AZD2014 (dual mTORC1/2 inhibitor), indicating a skew towards mTORC1 sensitivity with more aggressive leukemic disease. Rapamycin efficiently targeted the translation elongation axis eEF2/eEF2K downstream of mTORC1, resulting in eEF2 inactivation through induction of eEF2T56 phosphorylation. Rapamycin treatment of primary CLL cells halted proliferation, modulated eEF2K/eEF2 phosphorylation and inhibited MCL1 expression. Our studies demonstrate that mTORC1 plays an essential role in leukemia progression in vitro and in vivo in our CLL mouse model, with evidence for increased rapamycin sensitivity in aggressive secondary CLL transplants. Furthermore, the suppression of translation elongation through inactivation of eEF2 may offer a novel therapeutic target for blocking CLL progression.

Germinal centers (GCs) are essential sites for the production of high-affinity antibody secreting plasma cells (PCs) and memory-B cells (MBCs), which form the framework of vaccination. Affinity maturation and permissive selection in GCs are key for the production of PCs and MBCs, respectively. For these purposes, GCs positively select “fit” cells in the light zone of the GC and instructs them for one of three known B cell fates: PCs, MBCs and persistent GC-B cells as dark zone entrants. In this review, we provide an overview of the positive selection process and discuss its mechanisms and how B cell fates are instructed.

Significance Light zone (LZ) B cells are selected in germinal centers (GCs) in a cMyc-dependent manner, before dark zone (DZ) migration. Antigen affinity of B cells is enhanced during GC responses, and some differentiate into plasmablasts or memory B cells (MBCs). Currently, GC selection is presumed as a competitive affinity-dependent process. This cannot explain retention of GC B cells with varied affinities. We identified cMyc+ LZ B cell subpopulations enriched with either higher-affinity plasmablast precursors or lower-affinity MBC precursors and future DZ entrants, which diverged soon after permissive selection. Future DZ entrants’ affinity was enhanced through preferential proliferation of higher-affinity cells, and lower-affinity cells were retained in GCs. These findings elucidate mechanistically how GC selection ensures clonal diversity for broad protection., Affinity maturation depends on how efficiently germinal centers (GCs) positively select B cells in the light zone (LZ). Positively selected GC B cells recirculate between LZs and dark zones (DZs) and ultimately differentiate into plasmablasts (PBs) and memory B cells (MBCs). Current understanding of the GC reaction presumes that cMyc-dependent positive selection of LZ B cells is a competitive affinity-dependent process; however, this cannot explain the production of GC-derived lower-affinity MBCs or retention of GC B cells with varied affinities. Here, by combining single-cell/bulk RNA sequencing and flow cytometry, we identified and characterized temporally and functionally distinct positively selected cMyc+ GC B cell subpopulations. cMyc+ LZ B cell subpopulations enriched with either higher- or lower-affinity cells diverged soon after permissive positive selection. The former subpopulation contained PB precursors, whereas the latter comprised less proliferative MBC precursors and future DZ entrants. The overall affinity of future DZ entrants was enhanced in the LZ through preferential proliferation of higher-affinity cells. Concurrently, lower-affinity cells were retained in GCs and protected from apoptosis. These findings redefine positive selection as a dynamic process generating three distinct B cell fates and elucidate how positive selection ensures clonal diversity for broad protection.

The parasitic worm product, ES-62 protects against collagen-induced arthritis, a mouse model of rheumatoid arthritis (RA) by suppressing the synovial fibroblast (SF) responses perpetuating inflammation and driving joint destruction. Such SF responses are shaped during disease progression by the inflammatory microenvironment of the joint that promotes remodelling of their epigenetic landscape, inducing an “aggressive” pathogenic SF phenotype. Critically, exposure to ES-62 in vivo induces a stably imprinted “safe” phenotype that exhibits responses more typical of healthy SFs. Surprisingly however, DNA methylome analysis reveals that rather than simply preventing the pathogenic rewiring of SFs, ES-62 induces further epigenetic remodelling, including targeting genes associated with ciliogenesis and differentiation, to program a distinct “protective” phenotype. Such unique behaviour signposts potential DNA methylation signatures predictive of pathogenesis and its resolution and hence, candidate mechanisms by which novel therapeutic interventions could prevent SFs from perpetuating joint inflammation and destruction in RA.

Memory B cells (MBCs) are key for protection from reinfection. However, it is mechanistically unclear how germinal center (GC) B cells differentiate into MBCs. MYC is transiently induced in cells fated for GC expansion and plasma cell (PC) formation, so-called positively selected GC B cells. We found that these cells coexpressed MYC and MIZ1 (MYC-interacting zinc-finger protein 1 [ZBTB17]). MYC and MIZ1 are transcriptional activators; however, they form a transcriptional repressor complex that represses MIZ1 target genes. Mice lacking MYC–MIZ1 complexes displayed impaired cell cycle entry of positively selected GC B cells and reduced GC B cell expansion and PC formation. Notably, absence of MYC–MIZ1 complexes in positively selected GC B cells led to a gene expression profile alike that of MBCs and increased MBC differentiation. Thus, at the GC positive selection stage, MYC–MIZ1 complexes are required for effective GC expansion and PC formation and to restrict MBC differentiation. We propose that MYC and MIZ1 form a module that regulates GC B cell fate.

Retrovirus-mediated gene transfer has become a powerful tool to investigate roles of specific molecules in B cells, due to its efficiency and expeditiousness. This technology is applicable to activated B cells in order to determine effects of a gene of interest during germinal center (GC) reactions in combination with adoptive transfer. To achieve this, B cells derived from SW

Thymus-derived regulatory T (tTreg) cells are key to preventing autoimmune diseases, but the mechanisms involved in their development remain unsolved. Here, we show that the C-type lectin receptor CD69 controls tTreg cell development and peripheral Treg cell homeostasis through the regulation of BIC/microRNA 155 (miR-155) and its target, suppressor of cytokine signaling 1 (SOCS-1). Using Foxp3-mRFP/cd69 or Foxp3-mRFP/cd69 reporter mice and short hairpin RNA (shRNA)- mediated silencing and miR-155 transfection approaches, we found that CD69 deficiency impaired the signal transducer and activator of transcription 5 (STAT5) pathway in Foxp3 cells. This results in BIC/miR-155 inhibition, increased SOCS-1 expression, and severely impaired tTreg cell development in embryos, adults, and Rag2 γc hematopoietic chimeras reconstituted with cd69 stem cells. Accordingly, mirn155 mice have an impaired development of CD69 tTreg cells and overexpression of the miR-155-induced CD69 pathway, suggesting that both molecules might be concomitantly activated in a positive-feedback loop. Moreover, in vitro-inducible CD25 Treg (iTreg) cell development is inhibited in Il2rγ/cd69 mice. Our data highlight the contribution of CD69 as a nonredundant key regulator of BIC/miR-155-dependent Treg cell development and homeostasis., Spanish Ministry of Economy and Competitiveness (SAF2013-44857-R to M.L.T.); grant INDISNET 01592006 from the Comunidad de Madrid to P.M. and F.S.-M.; and grants from the Instituto de Salud Carlos III (PI-FIS-2016-9488 to P.M.); the CIBER de Enfermedades Cardiovasculares to F.S.-M. and P.M.; and the Fundació La Marató TV3 (20152330 31)

Retrovirus-mediated gene transfer has become a powerful tool to investigate roles of specific molecules in B cells, due to its efficiency and expeditiousness. This technology is applicable to activated B cells in order to determine effects of a gene of interest during germinal center (GC) reactions in combination with adoptive transfer. To achieve this, B cells derived from SWHEL mice expressing hen egg lysozyme (HEL)-specific B cell receptors (BCR) are stimulated with HEL antigen in vivo and then with anti-CD40 antibody ex vivo. These cells are then transduced with a retrovirus allowing bicistronic co-expression of a gene of interest and GFP, enabling differentiation of transgene positive cells. The retrovirally transduced cells are then adoptively transferred into immunized CD45.1+ congenic recipient mice, to enable differentiation between donor and host cells. This chapter describes methods for (1) activation of HEL-specific mature B cells, (2) retroviral transduction of the activated B cells, (3) adoptive transfer of the cells into recipients, and (4) analysis of the resultant mice by flow cytometry.

Lu et al. disrupt the interaction between miR-155 and the transcription factor PU.1 by specifically removing miR-155–binding site from PU.1 mRNA in mice. They show that this interaction is required for plasma cell formation and extrafollicular response to immunization in vivo., A single microRNA (miRNA) can regulate the expression of many genes, though the level of repression imparted on any given target is generally low. How then is the selective pressure for a single miRNA/target interaction maintained across long evolutionary distances? We addressed this problem by disrupting in vivo the interaction between miR-155 and PU.1 in mice. Remarkably, this interaction proved to be key to promoting optimal T cell–dependent B cell responses, a previously unrecognized role for PU.1. Mechanistically, miR-155 inhibits PU.1 expression, leading to Pax5 down-regulation and the initiation of the plasma cell differentiation pathway. Additional PU.1 targets include a network of genes whose products are involved in adhesion, with direct links to B–T cell interactions. We conclude that the evolutionary adaptive selection of the miR-155–PU.1 interaction is exercised through the effectiveness of terminal B cell differentiation.

SummaryMemory B cells are essential for generating rapid and robust secondary antibody responses. It has been thought that the unique cytoplasmic domain of IgG causes the prompt activation of antigen-experienced IgG memory B cells. To assess this model, we have generated a mouse containing IgG1 B cells that have never encountered antigen. We found that, upon challenge, antigen-experienced IgG1 memory B cells rapidly differentiated into plasma cells, whereas nonexperienced IgG1 B cells did not, suggesting the importance of the stimulation history. In addition, our results suggest that repression of the Bach2 transcription factor, which results from antigen experience, contributes to predisposition of IgG1 memory B cells to differentiate into plasma cells.

12 p.-6 fig.1 tab.Tomokatsu Ikawa, et al., In general, cell fate is determined primarily by transcription factors, followed by epigenetic mechanisms fixing the status. While the importance of transcription factors controlling cell fate has been well characterized, epigenetic regulation of cell fate maintenance remains to be elucidated. Here we provide an obvious fate conversion case, in which the inactivation of polycomb-medicated epigenetic regulation results in conversion of T-lineage progenitors to the B-cell fate. In T-cell-specific Ring1A/B-deficient mice, T-cell development was severely blocked at an immature stage. We found that these developmentally arrested T-cell precursors gave rise to functional B cells upon transfer to immunodeficient mice. We further demonstrated that the arrest was almost completely canceled by additional deletion of Pax5. These results indicate that the maintenance of T-cell fate critically requires epigenetic suppression of the B-lineage gene program., This work was supported in part by grants from the Japan Society for the Promotion of Science (24689042 to T.I.), the Japan Science and Technology Agency (T.I.), RIKEN Center for Integrative Medical Sciences (IMS) Young Chief Investigator program (T.I.), and the Kanae Foundation for the Promotion of Medical Science (T.I.).

During hematopoietic lineage development, hematopoietic stem cells sequentially commit toward myeloid or lymphoid lineages in a tightly regulated manner, which under normal circumstances is irreversible. However, studies have established that targeted deletion of the B-lineage specific transcription factor, paired box gene 5 (Pax5), enables B cells to differentiate toward other hematopoietic lineages, in addition to generating progenitor B-cell lymphomas. Our previous studies showed that subversion of protein kinase C (PKC)-α in developing B cells transformed B-lineage cells. Here, we demonstrate that PKC-α modulation in committed CD19+B lymphocytes also promoted lineage conversion toward myeloid, NK-, and T-cell lineages upon Notch ligation. This occurred via a reduction in Pax5 expression resulting from a downregulation of E47, a product of the E2A gene. T-cell lineage commitment was indicated by the expression of T-cell associated genes Ptcra, Cd3e, and gene rearrangement at the Tcrb gene locus. Importantly, the lineage-converted T cells carried Igh gene rearrangements reminiscent of their B-cell origin. Our findings suggest that modulation of PKC-α induces hematopoietic-lineage plasticity in committed B-lineage cells by perturbing expression of critical B-lineage transcription factors, and deregulation of PKC-α activity/expression represents a potential mechanism for lineage trans-differentiation during malignancies.

Death-associated protein kinase (DAPK) is a pro-apoptotic serine/threonine protein kinase that is dysregulated in a wide variety of cancers. The mechanism by which this occurs has largely been attributed to promoter hypermethylation, which results in gene silencing. However, recent studies indicate that DAPK expression can be detected in some cancers, but its function is still repressed, suggesting that DAPK activity can be subverted at a post-translational level in cancer cells. This review will focus on recent data describing potential mechanisms that may alter the expression, regulation or function of DAPK.

CLL (chronic lymphocytic leukaemia) is characterized by the clonal outgrowth of B-lymphocytes with the distinctive phenotype: CD19 hi CD5 + CD23 + IgM lo . These malignant B-cells accumulate in the PB (peripheral blood) and lymphoid organs, and are generally arrested at the G 0 /G 1 -phase of cell cycle and display a resistance to apoptosis. To date, most of the CLL research has been carried out using PB samples obtained from patients with established CLL, which have proved instrumental in characterizing the disease. However, while CLL cells appear to have a defect in apoptosis in vivo , they rapidly undergo apoptosis ex vivo , suggesting that CLL cells are dependent on microenvironmental signals to enhance cell survival. One approach used to define the cellular and molecular events that govern CLL has been the development of murine models that replicate the human disease. As well as providing a deeper understanding of the potential triggers for CLL, these models provide preclinical in vivo systems to test novel therapies. The focus of the present review will be to highlight the recent advances in the development of mouse models for CLL.

The production of high-affinity antibodies by B cells is essential for pathogen clearance. Antibody affinity for antigen is increased through the affinity maturation in germinal centers (GCs). This is an iterative process in which B cells cycle between proliferation coupled with the acquisition of mutations and antigen-based positive selection, resulting in retention of the highest-affinity B cell clones. The posttranscriptional regulator microRNA-155 (miR-155) is critical for efficient affinity maturation and the maintenance of the GCs; however, the cellular and molecular mechanism by which miR-155 regulates GC responses is not well understood. Here, we utilized a miR-155 reporter mouse strain and showed that miR-155 is coexpressed with the proto-oncogene encoding c-MYC in positively selected B cells. Functionally, miR-155 protected positively selected c-MYC+ B cells from apoptosis, allowing clonal expansion of this population, providing an explanation as to why Mir155 deletion impairs affinity maturation and promotes the premature collapse of GCs. We determined that miR-155 directly inhibits the Jumonji family member JARID2, which enhances B cell apoptosis when overexpressed, and thereby promotes GC B cell survival. Our findings also suggest that there is cooperation between c-MYC and miR-155 during the normal GC response, a cooperation that may explain how c-MYC and miR-155 can collaboratively function as oncogenes.

Overwhelming evidence identifies the microenvironment as a critical factor in the development and progression of chronic lymphocytic leukemia, underlining the importance of developing suitable translational models to study the pathogenesis of the disease. We previously established that stable expression of kinase dead protein kinase C alpha in hematopoietic progenitor cells resulted in the development of a chronic lymphocytic leukemia-like disease in mice. Here we demonstrate that this chronic lymphocytic leukemia model resembles the more aggressive subset of chronic lymphocytic leukemia, expressing predominantly unmutated immunoglobulin heavy chain genes, with upregulated tyrosine kinase ZAP-70 expression and elevated ERK-MAPK-mTor signaling, resulting in enhanced proliferation and increased tumor load in lymphoid organs. Reduced function of PKCα leads to an up-regulation of PKCβII expression, which is also associated with a poor prognostic subset of human chronic lymphocytic leukemia samples. Treatment of chronic lymphocytic leukemia-like cells with the selective PKCβ inhibitor enzastaurin caused cell cycle arrest and apoptosis both in vitro and in vivo, and a reduction in the leukemic burden in vivo. These results demonstrate the importance of PKCβII in chronic lymphocytic leukemia-like disease progression and suggest a role for PKCα subversion in creating permissive conditions for leukemogenesis.

A common method of determining the role of specific signaling molecules during lymphocyte development is to generate a transgenic mouse. This procedure, while informative, is time consuming, expensive and ultimately does not guarantee a defined answer. Here we present a protocol in which the in vivo effects of a gene of interest on both B and T lymphocyte development may be determined simultaneously in a relatively short time period. This is achieved by introducing a defined gene, such as a wild-type or mutated signaling molecule, into a lymphoid progenitor population by retroviral infection. The retrovirus generates a bicistronic message encoding the gene of interest and GFP, thus enabling identification of retrovirally transduced cells in subsequent lymphocyte lineages. The cells are then introduced into mice deficient for recombinase activating gene 1 (Rag-/- mice), thus allowing the development of donor-derived B and T lymphocytes in vivo. Using this technique, results can be obtained within 3-8 weeks.

B-cell chronic lymphocytic leukemia (B-CLL) is characterized by the accumulation of long-lived mature B cells with the distinctive phenotype CD19hi CD5+ CD23+ IgMlo, which are refractory to apoptosis. An increased level of apoptosis has been observed on treatment of human B-CLL cells with protein kinase C (PKC) inhibitors, suggesting that this family of protein kinases mediate survival signals within B-CLL cells. Therefore, to investigate the ability of individual PKC isoforms to transform developing B cells, we stably expressed plasmids encoding PKC mutants in fetal liver–derived hematopoietic progenitor cells (HPC) from wild-type mice and then cultured them in B-cell generation systems in vitro and in vivo. Surprisingly, we noted that expression of a plasmid-encoding dominant-negative PKCα (PKCα-KR) in HPCs and subsequent culture both in vitro and in vivo resulted in the generation of a population of cells that displayed an enhanced proliferative capacity over untransfected cells and phenotypically resemble human B-CLL cells. In the absence of growth factors and stroma, these B-CLL-like cells undergo cell cycle arrest and, consistent with their ability to escape growth factor withdrawal-induced apoptosis, exhibited elevated levels of Bcl-2 expression. These studies therefore identify a unique oncogenic trigger for the development of a B-CLL-like disease resulting from the subversion of PKCα signaling. Our findings uncover novel avenues not only for the study of the induction of leukemic B cells but also for the development of therapeutic drugs to combat PKCα-regulated transformation events. (Cancer Res 2006; 66(1): 527-34)

The NK cell receptor protein 1 (NKR-P1) (CD161) molecules represent a family of type II transmembrane C-type lectin-like receptors expressed predominantly by NK cells. Despite sharing a common NK1.1 epitope, the mouse NKR-P1B and NKR-P1C receptors possess opposing functions in NK cell signaling. Engagement of NKR-P1C stimulates cytotoxicity of target cells, Ca2+ flux, phosphatidylinositol turnover, kinase activity, and cytokine production. In contrast, NKR-P1B engagement inhibits NK cell cytotoxicity. Nonetheless, it remains unclear how different signaling outcomes are mediated at the molecular level. Here, we demonstrate that both NKR-P1B and NKR-P1C associate with the tyrosine kinase, p56lck. The interaction is mediated through the di-cysteine CxCP motif in the cytoplasmic domains of NKR-P1B/C. Disrupting this motif leads to abrogation of both stimulatory and inhibitory NKR-P1 signals. In addition, mutation of the consensus ITIM (LxYxxL) in NKR-P1B abolishes both its Src homology 2-containing protein tyrosine phosphatase-1 recruitment and inhibitory function. Strikingly, engagement of NKR-P1C on NK cells obtained from Lck-deficient mice failed to induce NK cytotoxicity. These results reveal a role for Lck in the initiation of NKR-P1 signals, and demonstrate a requirement for the ITIM in NKR-P1-mediated inhibition.

Protein kinase Calpha (PKCalpha) is a serine/threonine protein kinase that has been implicated in the regulation of a variety of cellular functions such as proliferation, differentiation and apoptosis in response to a diverse range of stimuli. In order to execute these biological events PKCalpha activity is modulated by, and functionally interacts with, a number of proto-oncogenes, therefore it is perhaps unsurprising that dysregulation of PKCalpha is associated with a diverse range of cancers. Recently, PKCalpha has become a target for a number of anti-cancer therapies. The purpose of this review is to describe how PKCalpha regulates key biological events, to gain an insight into how PKCalpha-mediated cellular transformation may occur. In this way, it may be possible to design therapeutic tools to combat cancers specifically associated with PKCalpha dysfunction.

The ABC half-transporter, ABCG2, is known to confer resistance to chemotherapeutic agents including indolocarbazole derivatives. MCF7 cells were introduced by either wild type ABCG2 (ABCG2-482R) or mutant ABCG2 (-482T), whose amino acid at position 482 is substituted to threonine from arginine, and their cross-resistance pattern was analyzed. Although this amino acid substitution seems to affect cross-resistance patterns, both 482T- and 482R-transfectants showed strong resistance to indolocarbazoles, confirming that ABCG2 confers resistance to them. For further characterization of ABCG2-mediated transport, we investigated indolocarbazole compound A (Fig. 1) excretion in cell-free system. Compound A was actively transported in membrane vesicles prepared from one of the 482T- transfectants and its uptake was supported by hydrolysis of various nucleoside triphosphates. This transport was inhibited completely by the other indolocarbazole compound, but not by mitoxantrone, implying that the binding site of mitoxantrone or the transport mechanisms for mitoxantrone is different from those of indolocarbazoles. These results showed that ABCG2 confers resistance to indolocarbazoles by transporting them in an energy-dependent manner.

During hematopoietic lineage development, hematopoietic stem cells sequentially commit toward myeloid or lymphoid lineages in a tightly regulated manner, which under normal circumstances is irreversible. However, studies have established that targeted deletion of the B-lineage specific transcription factor, paired box gene 5 (Pax5), enables B cells to differentiate toward other hematopoietic lineages, in addition to generating progenitor B-cell lymphomas. Our previous studies showed that subversion of protein kinase C (PKC)-α in developing B cells transformed B-lineage cells. Here, we demonstrate that PKC-α modulation in committed CD19(+) B lymphocytes also promoted lineage conversion toward myeloid, NK-, and T-cell lineages upon Notch ligation. This occurred via a reduction in Pax5 expression resulting from a downregulation of E47, a product of the E2A gene. T-cell lineage commitment was indicated by the expression of T-cell associated genes Ptcra, Cd3e, and gene rearrangement at the Tcrb gene locus. Importantly, the lineage-converted T cells carried Igh gene rearrangements reminiscent of their B-cell origin. Our findings suggest that modulation of PKC-α induces hematopoietic-lineage plasticity in committed B-lineage cells by perturbing expression of critical B-lineage transcription factors, and deregulation of PKC-α activity/expression represents a potential mechanism for lineage trans-differentiation during malignancies.

Death-associated protein kinase (DAPK) is a pro-apoptotic serine/threonine protein kinase that is dysregulated in a wide variety of cancers. The mechanism by which this occurs has largely been attributed to promoter hypermethylation, which results in gene silencing. However, recent studies indicate that DAPK expression can be detected in some cancers, but its function is still repressed, suggesting that DAPK activity can be subverted at a post-translational level in cancer cells. This review will focus on recent data describing potential mechanisms that may alter the expression, regulation or function of DAPK.

While advances have been made in the clinical treatment of chronic lymphocytic leukaemia (CLL) in recent years, it is still an incurable disease and therefore the identification of novel drug therapies is of paramount importance. Understanding the molecular mechanisms that govern the survival of CLL cells is fundamental in achieving this goal. A number of studies indicate that protein kinase C (PKC)- and phosphatidylinositol-3-kinase (PI3K)- mediated signalling pathways are central to CLL cell survival, and as such PKC has gained renewed interest as a potential drug target in CLL. This may be because it represents a closely-related family of ten protein kinases, which due to the redundancy that exists between isoforms offers an opportunity for the design of isoform specific inhibitors drugs that target leukaemic cells whilst showing reduced toxicity for normal cells. Indeed, PKC signalling pathways have already been considered as targets for specific anticancer drugs [1-3]. Therefore, this short review will focus on the effect of modulating PKC activity in CLL cells and explore whether targeting PKCs could represent a valid therapy for this leukaemia.

Delineating the molecular mechanisms that trigger the development of leukemic cells is of fundamental importance for the creation of therapeutic interventions to specifically target these rogue cells. B cell-chronic lymphocyte leukemia (B-CLL) is characterized by an accumulation of long-lived B-lineage cells with the distinctive phenotype: CD19hi, CD5+, CD23+, IgMlo, which are refractory to apoptosis and have undergone cell cycle arrest in the G0/G1 phase. The signaling events that control the development of B-CLL are not well defined. To address this, we generated a novel cellular system in which the spontaneous generation of B-CLL-like cells is achieved by stably expressing a plasmid-encoding dominant negative PKCα (PKCα-KR) in mouse FL-derived hematopoietic progenitor cells (HPCs) and then culturing these cells in a B cell generation system. The resultant cells bear hallmark characteristics of human B-CLL cells at the level of: (i) phenotype - CD19hi CD5+ CD23+ IgMlo; (ii) cell cycle phase - halted in G0/G1, and; (iii) resistance to apoptosis. Therefore, this system provides us with a unique opportunity to investigate the signaling events that control the initiation of B-CLL. In addition, we isolated a mouse B-CLL (mB-CLL) cell line during long-term culture of PKCα-KR-HPCs, which proliferates in the absence of growth factors or stroma, thus allowing us to investigate the signaling mechanisms that may be important for the expansion of B-CLL. In an effort to identify the key signaling pathways that are responsible for maintaining survival of B-CLL cells, we treated MIEV (vector control)-, PKCα-KR-HPCs and mB-CLL cells with pharmacological inhibitors against ERK-MAPK- and PI3K-mediated pathways, as these pathways have been implicated in mediating the survival of human B-CLL cells. We assessed the proportion of cells that were induced to undergo apoptosis (Annexin V binding) relative to control (DMSO-treated) cells. We found that treatment of MIEV- and PKCα-KR-HPCs with PI3K inhibitors (LY294002/ wortmannin: 1–50 μM) induced a significant increase in apoptosis compared with the control cells (MIEV: 4.7±0.14; PKCα-KR: 6.8±1.18 at 30 μM LY294002), while the level of apoptosis in these cells was unaffected upon treatment with MEK inhibitors (PD98059/U0126: 1–20 μM) compared with controls (MIEV: 0.9±0.1; PKCα-KR: 0.7±0.01 at 10 μM U0126). Conversely, treatment of mB-CLL cells with MEK1 inhibitors induced an increase in apoptosis compared with the control cells (7.7±4.21 at 10 μM U0126), while the level of apoptosis in these cells was unaffected upon treatment with PI3K inhibitors (1.6±0.84 at 30 μM LY294002). Our results suggest that the signaling pathways responsible for maintaining cell survival are differentially-regulated during the initiation vs. expansion phases of B-CLL. Additional studies of the phosphorylation/ activation status of ERK1/ERK2 in MIEV-, PKCα-KR-HPCs and mB-CLL cells revealed that while ERK1/ERK2 are unphosphorylated/ inactive in MIEV-HPCs, ERK1 only is constitutively phosphorylated in PKCα-KR-HPCs, while mB-CLL cells contain constitutively phosphorylated ERK1 and ERK2. The differential phosphorylation of ERK1/ERK2 may, at least in part, explain the differential regulation of survival in quiescent and proliferating B-CLL cells. Indeed, the activation of ERK-MAPK can occur in a PI3K-dependent or -independent fashion, depending on the cell type. Collectively, these studies suggest that PI3K- and ERK-MAPK signaling pathways are differentially-regulated to induce survival during the progression of B-CLL cells.