Academic literature on the topic 'Eμ-TCL1 mice'

Abstract Abstract 925 One of the most relevant prognostic factors in chronic lymphocytic leukemia (CLL) is expression of the protein tyrosine kinase ZAP-70. Typically, patients whose leukemic cells express ZAP-70 at 30–100% of the levels in normal T cells have aggressive disease, whereas patients whose leukemic cells do not express ZAP-70 or express only low levels of this protein have indolent disease. Previously, we and others demonstrated that ZAP-70 modulates B-cell receptor signaling and thus affects the capacity of the leukemic cells to respond to antigen stimulation. However, a direct link between an altered antigen response and CLL pathogenesis has still not been established and, more importantly, the question whether ZAP-70 directly contributes to the aggressiveness of the disease or is just a marker of aggressive CLL still remains to be answered. We have now addressed these issues by analyzing in vivo the impact of forced expression of ZAP-70 on the development and behavior of leukemias that arise in the Eμ-TCL1 transgenic (tg) mouse model of CLL. This model is characterized by the development of antigen-driven leukemias that resemble human CLL in many aspects but are always ZAP-70-negative. To force the expression of ZAP-70 in TCL1 leukemias, we generated two tg lines with targeted expression of ZAP-70 in the B cell compartment (ZAP70high and ZAP70low) and crossed them with Eμ-TCL1 tg mice. B cells in ZAP70high tg mice express similar levels of ZAP-70 as normal mouse T cells, whereas the levels of ZAP-70 in B cells of ZAP70lowtg mice are approximately 10 times lower. Both cohorts of Eμ-TCL1/ZAP70 double tg mice developed characteristic TCL1 leukemias. Eμ-TCL1/ZAP70low tg mice developed leukemias with onset and rate of progression similar to their ZAP-70-negative littermates, indicating that low levels of ZAP-70 do not alter the development and behavior of the disease. Surprisingly, Eμ-TCL1/ZAP70high tg mice developed leukemias with an approximately 2 month delay compared to their ZAP-70-negative Eμ-TCL1 tg littermates, which was contrary to the expectation that high ZAP-70 expression will accelerate leukemia development. The delay in leukemia development was especially evident at 6 months of age, when leukemic cells could be detected in the PB of 77% (10/13) of Eμ-TCL1 tg mice and only 24% (4/17) of Eμ-TCL1/ZAP70hightg mice (P=0.011). Since ZAP-70 expression can affect the migratory and adhesion capacity of human CLL cells in vitro, we first investigated if the delayed appearance of leukemic cells in the PB of Eμ-TCL1/ZAP70high tg mice could be due to increased retention of the leukemic cells in the lymphoid tissues. Assessment of tumor burden in the spleen, peritoneal cavity (PC), bone marrow and PB of 7 months old mice showed that the number of tumor cells in each compartment was significantly lower in Eμ-TCL1/ZAP70hightg mice than their Eμ-TCL1 littermates, suggesting that the delay in leukemia appearance is not caused by increased tissue retention but rather by reduced tumor growth. To investigate if ZAP-70 impairs tumor growth by affecting proliferation, we performed in vivo BrdU incorporation analysis of leukemic cells from spleen and PC of Eμ-TCL1 and Eμ-TCL1/ZAP70high tg mice. Spleen and PC samples were analyzed because they are the major sites of leukemia proliferation in Eμ-TCL1 tg mice. Interestingly, while the percentage of proliferating leukemic cells in the spleens of Eμ-TCL1 and Eμ-TCL1/ZAP70high tg mice was similar (mean % of BrdU+ cells ±SD: 6.81 ±1.67 and 6.15 ±2.92, respectively; P=n.s.), the percentage of proliferating leukemic cells in the PC of Eμ-TCL1/ZAP70high tg mice was significantly lower (mean % of BrdU+cells ±SD: 1.74 ±1.05 and 0.56 ±0.39, respectively; P=0.024). In summary, this study shows that ZAP-70 expression, per se, is unable to accelerate leukemia development and progression in an established in vivo model of CLL and suggests that ZAP-70 is not directly responsible for the greater disease severity in the poor prognosis subset of CLL. In addition, this study reveals that ZAP-70 in certain tissue environments can function as a negative regulator of leukemic cell proliferation, contrary to the widespread perception of ZAP-70 as a positive regulator of leukemic cell responses. Disclosures: No relevant conflicts of interest to declare.

Abstract Introduction Inhibition of Brd4, a bromodomain and extra-terminal (BET) protein, results in antiproliferative effects and terminal differentiation in the MYC-driven B-cell malignancies multiple myeloma and Burkitt's lymphoma by selectively repressing MYC. Elevated MYC expression correlates with progression of B-cell chronic lymphocytic leukemia (B-CLL) marking Brd4 as a potential therapeutic target. 17p deletion or somatic TP53 mutations are the poorest prognostic factors in B-CLL, resulting refractoriness to conventional therapy. The recent identification of a Brd4-interacting protein as a validated target in CLL led us to evaluate the potential of BET inhibition, using jq1, for the treatment of B-CLL. Results Jq1 remarkably reduced proliferation of the immortalized human B-CLL cell lines, Mec-1, Mec-2, and WaC3 (IC50 values: 1.12, 0.68, and 0.64 μM, respectively). These results were validated in 13 frozen and 7 fresh primary whole peripheral blood B-CLL samples (median EC50 0.66 μM [range, 0.051 to 2.169 μM]) using a FACS-based proprietary platform (Vivia Biothech). Eμ-TCL1 (n=13) & Eμ-TCL1:p53R172H/+ (n=9) mice treated with intraperitoneal (IP) injections of jq1 at 50 mg/kg/day for 21 consecutive days had a significant reduction in lymphocytes and white blood cells (WBC) (with excellent tolerance and no off-target effects on hemoglobin level or platelet counts) compared to vehicle-treated mice. The median lymphocyte count at baseline and after 2 weeks of jq1 therapy was 13.4 & 5.92 x10^3/μL (p=0.008) for Eμ-TCL1 mice and 13.2 & 7.2 x10^3/μL (p=0.009) for Eμ-TCL1:p53R172H/+ mice. jq1 therapy significantly prolonged survival in both Eμ-TCL1 (428 vs 377 days, p=0.05) and Eμ-TCL1:p53R172H/+ (382.5 vs 300 days, p=0.0003) mice compared to mice receiving vehicle. FACs analysis was performed on blood taken from Eμ-TCL1 (n=5) and Eμ-TCL1:p53R172H/+ (n=6) mice at baseline and after 7 daily IP jq1 doses. Eμ-TCL1:p53R172H/+ mice showed a significant reduction in CD5+/CD19+ lymphocytes (mean difference, 14.37 [95% CI, 2.4 to 26.3]; p=0.03), which was not observed in Eμ-TCL1 mice. Cell cycle analysis of cultured Eμ-TCL1 splenocytes treated with jq1 showed a significant increase (p<0.0005) in the number of sub-G1 cells compared to untreated splenocytes, regardless of p53 status. Sensitivity against fludarabine was also tested in these samples and we show that 10μM fludarabine did not elicit significant changes in the number of cells in sub-G1 compared to untreated cells but a significant difference (p=0.009) was observed between splenocytes treated with 10μM fludarabine and 1μM jq1. In depth analyses of RNA sequencing data of splenocytes from 10 matched-pairs samples (5 Eμ-TCL1 & 5 Eμ-TCL1:p53R172H/+) treated in culture with 0 or 1μM jq1 for 24 hours are currently underway to identify BET targets in B-CLL, but preliminary gene expression analysis reveal that the sensitivity of B-CLL to BET inhibition is not Myc-dependent despite elevated Myc levels. Notably, c-Fos was one of the most significant differentially expressed genes, regardless of p53 status. Interestingly, AP-1 complex, a heterodimer of c-Fos and c-Jun, is inhibited by direct interaction with Tcl-1. We are currently validating these findings in additional samples and performing functional assays to gain a better understanding of BET targets in B-CLL. Conclusion BET bromodomain inhibition markedly reduced proliferation in both human and mouse B-CLL samples. Furthermore, the loss of wild-type p53 activity did not desensitize CLL cells to jq1, which was also effective at inducing massive cell death in fludarabine resistant CLL cells, regardless of p53 status. Mice receiving jq1 exhibited a significant reduction in leukemic burden and prolonged survival compared to control mice, with a more pronounced effect observed in mice with mutant p53. These data demonstrate the potent effect of BET inhibition in B-CLL and more importantly, provide evidence as an effective treatment for aggressive forms of B-CLL with 17p deletion or TP53 mutation or chemoresistant refractory disease. Disclosures: Primo: Vivia Biotech: Employment. Rojas:Vivia Biotech: Employment. Martinez:Vivia Biotech: Employment. Bradner:Tensha Therapeutics: Dr. Bradner is a scientific founder of Tensha Therapeutics, which is developing drug-like derivatives of the JQ1 bromodomain inhibitor as cancer therapeutics, through a license from the Dana-Farber Cancer Institute. Other. Ballesteros:Vivia Biotech: Employment.

Abstract Abstract 181 Studies conducted over the past decade have revealed a strong association between the mutational status of the immunoglobulin heavy-chain variable region (IGHV) genes and clinical course in patients with chronic lymphocytic leukemia (CLL). In patients with aggressive CLL, the leukemic cells typically express B cell receptors (BCRs) encoded by unmutated IGHV genes, whereas these genes are most often mutated in leukemic cells from patients with indolent disease. The mutational status of the IGHV genes reflects features of the antigen, such as antigen structure, form, presentation and affinity, indicating that the difference in the clinical course between IGHV-unmutated and IGHV-mutated CLL could be due to recognition of different types of antigens. In line with this possibility, recent studies have shown that IGHV-unmutated CLL (U-CLL) cells frequently express polyreactive BCRs that bind with low affinity to both microbial antigens and autoantigens translocated or exposed on apoptotic cells, whereas such reactivity is infrequent in IGHV-mutated CLL (M-CLL). To further explore the possibility that the clinical course in CLL is determined by the availability of particular types of antigenic stimuli, we investigated the impact of different antigen/BCR interactions on leukemia development and behavior in the Eμ-TCL1 transgenic mouse model of CLL. We initially established three cohorts of Eμ-TCL1 transgenic mice that expressed transgenic BCRs with different antigen specificity. Two of these cohorts expressed low-affinity unmutated transgenic BCRs reactive with the antigens phosphatidylcholine (PtC) and Sm (IgPtC and IgSm, respectively), whereas the third cohort expressed a high-affinity mutated transgenic BCR (IgHEL) specific for the antigen hen egg lysozyme (HEL). Of note, Sm is a ribonucleoprotein complex that is translocated to the surface of apoptotic cells and has been shown to be recognized by certain human U-CLL BCRs, whereas PtC is a cell membrane component that is exposed on senescent red blood cells and gut bacteria. Because no data are currently available regarding the reactivity of the M-CLL BCRs, we subdivided the cohort of Eμ-TCL1/IgHEL double transgenic mice into four additional cohorts. These included a cohort without antigen (Eμ-TCL1/IgHEL), a cohort in which HEL was provided as a foreign antigen (Eμ-TCL1/IgHEL double transgenic mice repetitively immunized with particles coated with HEL and CpG oligonucleotides), a cohort in which HEL was provided as a soluble autoantigen (Eμ-TCL1/IgHEL/sHEL triple transgenic mice) and a cohort in which HEL was provided as a membrane-bound autoantigen exposed on apoptotic cells (Eμ-TCL1/IgHEL/mHEL-KK triple transgenic mice). Each cohort consisted of 12–14 animals, of which at least 8 have been followed for >1 year. Animals from all cohorts developed CD5-positive B cell leukemias, but only in Eμ-TCL1/IgSm and Eμ-TCL1/IgPtC mice the leukemic cells expressed a transgenic BCR. In Eμ-TCL1/IgHEL mice the leukemias were always derived from the small percentage of B cells that express an endogenous BCR, whereas B cells that express the transgenic IgHEL BCR were never transformed. Interestingly, leukemia development and progression was more rapid in Eμ-TCL1/IgPtC than Eμ-TCL1/IgSm transgenic mice (7/14 at 6 months of age and 2/10 at 8 months of age, respectively). Since PtC is expressed as both a foreign- (gut flora) and self- (senescent red blood cells) antigen, we investigated whether suppression of gut flora will affect the growth of adoptively transferred Eμ-TCL1/IgPtC leukemias. Pretreatment of syngeneic recipient mice with a three-week course of broad-spectrum antibiotics significantly delayed leukemia growth, suggesting that PtC is more potent in driving the expansion of the leukemic clone when expressed as a foreign than self antigen. To summarize, these data demonstrate that U-CLL can be induced by both microbial antigens and autoantigens exposed on apoptotic cells, including autoantigens that are recognized by human CLL cells, such as Sm. In contrast, M-CLL can not be induced by chronic or repetitive antigen stimulation, regardless whether the antigen is provided as a foreign antigen, as a soluble autoantigen, or as a membrane-bound autoantigen exposed on apoptotic cells. Collectively, these data suggest that the mechanisms that drive U-CLL and M-CLL are different and indicate that only U-CLL is an antigen-driven disease. Disclosures: No relevant conflicts of interest to declare.

Abstract Wnt/Fzd signaling is known to play a key role in development, tissue specific stem cell maintenance, and tumorigenesis, particularly through the canonical pathway involving stabilization of β-catenin. We have previously shown that Fzd9−/− mice exhibit a decrease in pre-B cells at a stage when self-renewing division is occurring in preference to further differentiation, prior to light chain immunoglobulin recombination. To determine whether pathologic usurpation of this pathway plays a role in B cell leukemogenesis, we examined the expression of Wnt/Fzd pathway genes in the Eμ-TCL1 mouse model of chronic lymphocytic leukemia (CLL). We find that in the course of leukemogenesis, the expression of Wnt16, Wnt10b, Fzd1, and most dramatically, Fzd6, are progressively upregulated in the transformed CD5+ B cells of these mice, as are β-catenin protein levels. Fzd6 expression is increased an average of 35-fold in tumor B cells in comparison to CD5- normal B cells in the same mouse. In 3 animals we were able to compare oligoclonal, preleukemic CD5+ B cells with monoclonally transformed CD5+ B cells and noted between 4 and 340 fold incremental increases in Fzd6 expression. Elimination of Fzd6 gene expression by crossing Eμ-TCL1 into Fzd6−/− mice significantly delays or eliminates development of CLL in this model, while crossing into Fzd9−/− mice has no effect. Greater than 50% of Fzd6+/+ X Eμ-TCL1 mice have evidence of CLL in the peripheral blood at 5 months of age, while in Fzd6−/− X Eμ-TCL1 mice, this occurs at 8 months (p < 0.001). Even at 1 yr. of age, >30% of Fzd6−/− X Eμ-TCL1 mice remain disease free while >95% of Fzd6+/+ X Eμ-TCL1 mice have leukemia at 7 months of age. Tumors that do arise in Fzd6−/− mice are morphologically similar to the usual Eμ-TCL1 CLL cells, but do not show upregulation of β-catenin protein by flow cytometry, suggesting that Fzd6 is responsible for this aberrant expression. Further, rather than upregulating Wnt16, Wnt10b, and Fzd1, these genes are down-modulated in tumor cells lacking Fzd6, suggesting a balance between positive and negative signals in CLL B cells mediated by the Wnt/Fzd pathway with Fzd6 acting as an oncogene and other family members potentially as tumor suppressors. We and others have noted increases in LEF-1, Wnt10b, Wnt16, and Fzd3 in human CLL B cells as well, mirroring the findings in the mouse model. Our findings suggest that the self-renewal signals mediated by Wnt/Fzd that are enlisted during B cell development may be pathologically reactivated in the neoplastic transformation of mature B cells; and that agents targeting specific members of the beta-catenin signaling pathway may have therapeutic impact that could be tested in the Eμ-TCL1 model.

In Chronic Lymphocytic Leukemia (CLL), mature CD5+ B cells accumulate in lymphoid organs such as bone marrow and lymph nodes where they proliferate and expand within localized proliferation centers. In vitro and in vivo data suggest that survival and proliferation of CLL cells within proliferation centers may be also dependent on microenvironmental interactions originating from the surrounding cellular elements (e.g. monocyte-derived nurse-like cells, mesenchymal stromal cells, or CD4+ T lymphocytes), that deliver both membrane-bound and soluble signals to CLL cells. In particular, the role of CD4+ T cells in vivo is less defined and data in animal models are conflicting as they appear to sustain CLL clone expansion and survival through CD40L-CD40 interactions, though in approximately 40% of patients with CLL, aggressive leukemic clones appear to be independent of CD40 stimulation. We aimed at clarifying the role of CD4+ T lymphocytes taking advantage of the Eμ-TCL1 mouse model, which develops a disease that mimics aggressive, human CLL. To this aim, we generated genetically modified Eμ-TCL1 mice lacking either CD4+ T cells (TCL1+/+AB0), CD40 (TCL1+/+CD40-/-), or CD8+ T cells (TCL1+/+TAP-/-). In these mice, disease appearance and progression were monitored in lymphoid organs and blood by flow cytometry and immunohistochemistry analyses. Findings were confirmed by adoptive transfer of leukemic clones into mice either lacking CD4+ T cells, or CD40L, or treated with monoclonal antibodies depleting selected T cell populations, or blocking CD40L-CD40 interactions. Interestingly, we observed that CLL clones did not expand in mice either lacking or depleted of CD4+ T cells, thus confirming that CD4+ T cells are essential for CLL development in Eμ-TCL1 mice. On the contrary, in TCL1+/+TAP-/- mice, lacking CD8+ T cells, disease progression was accelerated, suggesting an anti-tumor activity exerted by this subset of T cells. Specificity of CD4+ T cells was marginal for CLL development, as leukemic clones developed regularly in transgenic mice whose CD4+ T cells had TCR with CLL-unrelated specificities. Similarly, TCL1+/+CD40-/- mice developed frank CLL with no differences compared to controls, as well as leukemic clones expanded when transferred into wild type mice treated with monoclonal antibodies blocking CD40, or into CD40L-/- mice, suggesting a dispensable role for CD40/CD40L stimulation in the development of murine CLL. Analysis of peritoneal fluid, spleen, lymph nodes and bone marrow showed similar CLL development in Eμ-TCL1 and TCL1+/+CD40-/- mice. In conclusion, our data demonstrates that CD8+ and CD4+ T cells exert opposite roles in CLL: CD8+ T cells restrain CLL progression, whereas CD4+ T cells support the expansion of CLL clones in Eμ-TCL1 mice through CD40-indipendent, and apparently non-cognate mechanisms. Further studies are warranted to dissect the nature of the molecules, either soluble or membrane-bound, responsible for the interactions occurring between CD4+ T cells and CLL B cells fueling the onset and expansion of CLL cells. Disclosures Ghia: AbbVie: Consultancy, Honoraria, Research Funding; Acerta/AstraZeneca: Consultancy, Honoraria; ArQule: Consultancy, Honoraria; BeiGene: Consultancy, Honoraria; Dynamo: Consultancy, Honoraria; Gilead: Consultancy, Honoraria, Research Funding; Janssen: Consultancy, Honoraria, Research Funding; Juno/Celgene: Consultancy, Honoraria; Sunesis: Consultancy, Honoraria, Research Funding; Novartis: Research Funding; Pharmacyclics LLC, an AbbVie Company: Consultancy.

Abstract The most common human leukemia is B-cell chronic lymphocytic leukemia (B-CLL), which is characterized by a progressive accumulation of abnormal B-lymphocytes in blood, bone marrow and secondary lymphoid organs. Typically disease progression is slow, but as the number of leukemic cells increases, they interfere with the production of other important blood cells, causing the patients to be in an immunosuppressive state. To study the basis of this immunoregulation, we used cells from the transgenic Eμ-Tcl1 mouse, which spontaneously develop B-CLL due to a B-cell specific expression of the oncogene, Tcl1. Previously we showed that Eμ-Tcl1 CLL cells constitutively produce an anti-inflammatory cytokine, IL-10. Here we studied the role of IL-10 in CLL cell survival in vitro and the development of CLL in vivo. We found that neutralization of IL-I0 using anti-IL-10 antibodies or blocking the IL-10 receptor (IL-10R) using anti-IL-10R antibodies did not affect the survival of CLL cells in vitro. On the other hand, adoptively transferred Eμ-Tcl1 cells grew at a slower rate in IL-10R KO mice vs. wild type (WT) mice. There was a significant reduction in CLL cell engraftment in the spleen, bone marrow, peritoneal cavity and liver of the IL-10R KO compared to WT mice. Further studies revealed that IL-10 could be playing a role in the tumor microenvironment possibly by affecting anti-tumor immunity. This was seen by a reduction in the activation of CD8+ T cells as well as a significantly lower production of IFN-γ by CD4+ T cells purified from CLL-injected WT mice compared to those purified from CLL-injected IL-10R KO mice. These studies demonstrate that CLL cells suppress host anti-tumor immunity via IL-10 production. This led us to investigate possible mechanisms by which IL-10 is produced. We found a novel role of B-cell receptor (BCR) signaling pathway in constitutive IL-10 secretion. Inhibition of Src or Syk family kinases reduces the constitutive IL-10 production by Eμ-Tcl1 cells in a dose dependent manner. In addition, we found that Eμ-Tcl1 CLL cells exhibit clonal variation in their IL-10 production in response to BCR cross-linking. Further studies are being performed to understand the mechanisms by which BCR signaling affects IL-10 production. Disclosures No relevant conflicts of interest to declare.

Abstract Chronic Lymphocytic Leukemia (CLL), the most common adult leukemia in the western world, is characterized by accumulation of clonally expanded CD5+CD19+ B lymphocytes in blood and secondary lymphoid organs with impaired apoptotic mechanisms. Prostate apoptosis response-4 (Par-4) is a pro-apoptotic tumor suppressor protein, which is silenced by promoter methylation in more than 30% of all cancers. It is also secreted and induces apoptosis selectively in many types of cancer cells but not in normal cells. Here we characterized the role of Par-4 in CLL cells using a murine model. The Eμ-Tcl1 mouse serves as an excellent model to study CLL as they develop a CLL like disease by 9–13 months of age, due to B cell specific over-expression of the oncogene, T cell Leukemia 1 (Tcl1). Adoptive transfer of primary CD5+CD19+ CLL cells from the Eμ-Tcl1 CLL mice into recipient syngeneic mice leads to the development of a CLL like disease within 3–5 weeks of transfer. Surprisingly Eμ-Tcl1 CLL cells constitutively express more Par-4 than normal B-1 or B-2 cells in mice. These CLL cells also secrete Par-4 which is functional in being cytotoxic to prostate cancer cells. We showed that Eμ-Tcl1 CLL cells have constitutively active B-cell receptor signaling and that inhibition of BCR signaling causes a decrease in Par-4 expression and increases apoptosis. Interestingly, we have found that shRNA mediated knockdown of Par-4 in human CLL cell lines, results in their reduced growth. These results suggest that intrinsic Par-4 may play a pro-survival rather than pro-apoptotic role in CLL. We are currently investigating the mechanisms underlying this novel role of Par-4 and regulation of its expression in CLL cells using CRISPR system to knockout Lyn and Btk in CLL cells.

Abstract Abstract 313 Background: We have previously demonstrated that CD4 and CD8 T-cells from CLL patients show profound dysfunctions in multiple gene pathways, including the actin cytoskeleton, which impairs the formation of functional immunologic synapses between T cells and APCs. Functional screening assays on Mec-1 cells have identified CD200, CD270, CD274, and CD276 as inhibitory ligands which induce impaired actin synapse formation in both allogeneic and autologous T cells. We also demonstrated that the Eμ-TCL1 transgenic mouse model of CLL closely resembles the T-cell defects observed in humans, validating it as a valuable preclinical tool to examine changes in the microenvironment alongside the development of leukaemia. The aim of the current study is to investigate the role of CD200, CD270, CD274, and CD276 in the Eμ-TCL1 model. Methods: We used multiparameter flow cytometry to establish the expression of inhibitory ligands on CD19+/CD5+ unpurified splenocytes from Eμ-TCL1 mice on both the C57Bl/6 (B6) and the C3HB6-F1 background and compared this to unpurified splenocytes from age matched wild-type (WT) controls of the respective coisogenic strain. Results: A total of 19 leukemic Eμ-TCL1 (n=10 C57Bl/6 and n=9 C3HB6-F1 background) and 11 WT mice (n=6 C57Bl/6 and n=5 C3HB6-F1 background) were examined. CD19+/CD5+ CLL cells constituted 92% (range 62–97%) of the DAPI-negative lymphocyte population. On CD19+/CD5+ CLL cells, CD274 (mean 98% ± SEM 0.4) and CD200 (mean 84% ± SEM 2.9 were uniformly strongly expressed, while CD270 (mean 74% ± SEM 4.7) and CD276 (mean 50% ± SEM 6.6) showed a weaker and more diverse expression, with no significant differences between the two backgrounds (all p>.05). Similar expression patterns were observed in Eμ-TCL1 mice with spontaneously occurring CLL and transplanted transgenic mice, with no differences between spontaneous and induced CLL (all p>.05). We then compared transgenic CD19+/CD5+ CLL cells to the WT CD19+ and the WT CD19+/CD5+ B1a-like cell population. Eμ-TCL1 CLL splenocytes showed a significant higher expression of CD274 and CD276 compared to expression on WT CD19+ (p<.0001, p=.00349) splenocytes. When compared to WT B1a-like splenocytes, only CD274 was significantly higher expressed (p<.0001). To clarify the impact of genetic strain, B6 and C3HB6-F1 were investigated separately: transgenic mice on the B6 background showed significantly higher expression of CD274 compared to WT B6 CD19+ (p=.0015) and WT B6 B1a-like (p<.0001) splenocytes. In contrast, transgenic mice on the C3HB6-F1 background showed a significant higher expression of CD274 and CD276 compared to WT CD19+ (p=.0002, p=.00354) and WT B1a-like (p=.0005, p=.00384) splenocytes. These patterns substantiate differences of the expression of inhibitory ligands between the WT strains, but of note, these were not mirrored in TCL1 mice. In previous experiments, we used the Eμ-TCL1 model to investigate the polarization of F-actin and phosphotyrosine at the immune synapse between splenic autologous T-cells and APCs and subsequent effector function. Age-matched WT mice had a significantly higher accumulation than transgenic mice. To assess the functional role of inhibitory ligands, knock-down experiments using lentiviral shRNA and blocking antibodies are currently under way to assess if this restores immune synapse formation and T cell effector function in vivo. Conclusions: The inhibitory ligands CD200, CD270, CD274 and CD276 are expressed in vivo and appear to be of functional relevance for the anti-cancer immune response. They therefore represent attractive targets to restore T-cell effector function, which might be achieved by gene therapy approaches and blocking antibodies. Disclosures: Gribben: Celgene: Honoraria.

Abstract Chronic lymphocytic leukemia (CLL) represents 30% of adult leukemia. TCL1 is expressed in ∼ 90% of human CLL. Transgenic expression of TCL1 in murine B cells (Eμ-TCL1) results in mouse CLL. Here we show for the first time that the previously unexplored endoplasmic reticulum (ER) stress response is aberrantly activated in Eμ-TCL1 mouse and human CLL. This includes activation of the IRE-1/XBP-1 pathway and the transcriptionally up-regulated expression of Derlin-1, Derlin-2, BiP, GRP94, and PDI. TCL1 associates with the XBP-1 transcription factor, and causes the dysregulated expression of the transcription factors, Pax5, IRF4, and Blimp-1, and of the activation-induced cytidine deaminase. In addition, TCL1-overexpressing CLL cells manufacture a distinctly different BCR, as we detected increased expression of membrane-bound IgM and altered N-linked glycosylation of Igα and Igβ, which account for the hyperactive BCR in malignant CLL. To demonstrate that the ER stress-response pathway is a novel molecular target for the treatment of CLL, we blocked the IRE-1/XBP-1 pathway using a novel inhibitor, and observed apoptosis and significantly stalled growth of CLL cells in vitro and in mice. These studies reveal an important role of TCL1 in activating the ER stress response in support for malignant progression of CLL.

AbstractResults of heavy-water labeling studies have challenged the notion that chronic lymphocytic leukemia (CLL) represents an accumulation of noncycling B cells. We examined leukemia cell turnover in Eμ-TCL1 transgenic (TCL1-Tg) mice, which develop a CLL-like disease at 8 to 12 months of age. We found that leukemia cells in these mice not only had higher proportions of proliferating cells but also apoptotic cells than did nonleukemic lymphocytes. We crossed TCL1-Tg with BAFF-Tg mice, which express high levels of CD257. TCL1×BAFF-Tg mice developed CLL-like disease at a significantly younger age and had more rapid disease progression and shorter survival than TCL1-Tg mice. Leukemia cells of TCL1×BAFF-Tg mice had similar proportions of proliferating cells, but fewer proportions of dying cells, than did the CLL cells of TCL1-Tg mice. Moreover, leukemia cells from either TCL1×BAFF-Tg or TCL1-Tg mice produced more aggressive disease when transferred into BAFF-Tg mice than into wild-type (WT) mice. Neutralization of CD257 resulted in rapid reduction in circulating leukemia cells. These results indicate that the leukemia cells of TCL1-Tg mice undergo high levels of spontaneous apoptosis that is offset by relatively high rates of leukemia cell proliferation, which might allow for acquisition of mutations that contribute to disease evolution.

Chronic lymphocytic leukemia is an haematological malignancy characterized by the accumulation of CD5+CD19+ mature B cell clones in peripheral blood, spleen, lymph nodes and bone marrow (B-CLL). Leukemic cells accumulation is both dependent upon enhanced B cell receptor (BCR)-driven proliferation and defects in the apoptotic machinery. Additionally, alterations of surface chemokine receptors have been accounted for the enhanced accumulation and survival of CLL cells within the secondary lymphoid organs (SLOs). At present, treatment options for CLL patients include drugs targeting proteins participating in the BCR signaling pathway (i.e Ibrutinib, Idelalisib) and in the apoptotic process (i.e Venetoclax). However, relapsed or refractory CLL is still a clinical problem, and prolonged treatment with the above mentioned drugs leads to resistance occurrence as well as severe side effects, indicating that novel therapeutic options are needed. The adaptor protein p66Shc is a negative regulator of BCR-signaling and a pro-apoptotic protein whose loss in CLL B cells accounts for the extended survival of tumoral cells and poor prognosis. p66Shc has been also shown to control B cells trafficking by altering CXCR4 function, CCR7 and S1PR1 expression. The pathogenetic role of p66Shc in CLL was recently confirmed in the Eμ-TCL1 mouse model of CLL. Eμ-TCL1 mice lacking the p66Shc gene (Eμ-TCL1p66Shc-/- mice), showed indeed a more severe disease characterized by earlier onset and higher nodal and extranodal accumulation of leukemic cells compared with the Eμ-TCL1 mice. Hence, identification of compounds that up-regulate p66Shc expression and function in malignant B cells are likely to target cell proliferation, migration and apoptosis and may represent an appealing therapeutic option for CLL. In this study, we decided to investigate the molecular mechanism underlying the anti tumoral efficacy of a recently developed class of compounds, the pyrrolonaphthoxazepines (PNOXs), which have been demonstrated to be effective in vitro in several tumoral cells, including primary human CLL cells and to test, for the first time, these novel compounds in the murine model of human CLL, the Eμ-TCL1 mouse. Our results show that PNOXs restore p66Shc expression and activate transcription factor STAT4 in primary human CLL cells without affecting normal B cells. Accordingly, our data show that the recovery of p66Shc expression in human CLL cells directly correlates with the apoptosis rate induced by PNOXs. In agreement with human CLL, we found that PNOXs efficiently upregulate p66Shc and promote apoptosis in murine CLL cells. Moreover, we demonstrated that PNOXs were able to activate pro-apoptotic activity of p66Shc through a JNK dependent phosphorylation of Ser36 residue on p66Shc, suggesting that the anti-tumor effect of PNOXs in human CLL cells relies at least in part on its ability to first promote p66Shc expression and subsequently to foster its apoptotic function. PNOXs treatment of Eμ-TCL1 mice resulted in a significantly longer overall survival and in the reduction in tumor burden in the spleen and in the peritoneum. The powerful antitumor effect of PNOXs in vivo correlates with upregulation of S1PR1 and in the mobilization of leukemic cells from the spleen into the blood. Interestingly, treatment with PNOXs compounds on Eμ-TCL1p66Shc-/- mice highlighted that only one compound, PNOX-3, exerts a p66Shc-independent pro-apoptotic activity towards circulating CLL cells, while absence of p66Shc impairs the ability of PNOXs to mobilize CLL cells from the spleen to peripheral blood. Since accumulation of leukemic cells within secondary lymphoid organs is responsible for the enhanced survival of leukemic cells and drug resistance, our data indicate that PNOXs may represent a novel effective treatment for CLL by favouring the mobilization of leukemic cells from the protective tumor microenvironment of the SLOs into the blood and by promoting apoptosis through the upregulation of p66Shc.