TCL1 Mice Research Articles

Exome sequencing of the TCL1 mouse model for CLL reveals genetic heterogeneity and dynamics during disease development

The TCL1 mouse model is widely used to study pathophysiology, clonal evolution, and drug sensitivity or resistance of chronic lymphocytic leukemia (CLL). By performing whole exome sequencing, we present the genetic landscape of primary tumors from TCL1 mice and of TCL1 tumors serially transplanted into wild-type recipients to mimic clonal evolution. We show that similar to CLL patients, mutations in mice are frequently subclonal and heterogenous among different primary TCL1 mice. We further describe that this molecular heterogeneity mirrors heterogenous disease characteristics such as organ infiltration or CLL dependent T cell skewing. Similar to human CLL, we further observed the occurrence of novel mutations and structural variations during clonal evolution and found plasticity in the expansion of B cell receptor specific subclones. Thus, our results uncover that the genetic complexity, pathway dependence and clonal dynamics in mouse CLL are in relevant agreement to human CLL, and they are important to consider in future research using the TCL1 mouse for studying CLL.

Abstract 1964: Preclinical efficacy of the HSP90 inhibitor SNX-5422 in targeting C481S mutant BTK and ibrutinib resistant CLL

Abstract While chronic lymphocytic leukemia (CLL) is effectively treated by the Bruton's tyrosine kinase (BTK) inhibitor ibrutinib, patients that do relapse on ibrutinib (approximately 19%) have an extremely poor clinical outcome without further intervention. Before clinical diagnosis of relapse, signs of molecular relapse can be identified through deep sequencing for mutations in BTK (C481S) and the immediate downstream BTK target, PLCγ2. The BTK C481S mutation (which reduces ibrutinib binding) can be identified at frequencies as low as 5%, and expansion of this mutant close inevitably leads to clinical relapse. Therefore, identifying methods to target this mutant clone before clinical relapse represents a novel and potentially effective therapeutic strategy. HSP90 is a chaperone protein responsible for maintaining the stability of a large number of client proteins involved in cancer development. Cancer cells, particularly those reliant on a mutated oncogene, rely heavily on HSP90 for survival, making this an attractive clinical target. Here we have explored the preclinical efficacy of SNX-5422, a chemically unique, orally bioavailable HSP90 inhibitor, in ibrutinib resistant CLL. We determined that SNX-5422 can degrade C481S mutated BTK by utilizing BTK-null B-cell lines over-expressing wildtype or C481S BTK. Whereas ibrutinib reduces BTK activity in only the wildtype BTK transfected cell line, SNX-5422 degrades both wildtype BTK and C481S mutant BTK and impairs downstream signaling. In fact, C481S mutant BTK shows increased degradation compared to wildtype BTK suggesting that the C481S mutant BTK may rely on HSP90 more for stabilization. We next examined the in vivo efficacy of SNX-5422 in a mouse model of ibrutinib-resistant CLL. Leukemic splenocytes isolated at removal criteria from TCL1 mice continuously treated with ibrutinib were adoptively transferred into syngeneic WT B6 mice. Upon reaching 10% leukemia burden in the peripheral blood, the mice were randomized into 1 of 4 treatment groups: vehicle, SNX-5422 (50 mg/kg MWF), ibrutinib (continuous in drinking water), or SNX-5422 plus ibrutinib (concurrent). Mice treated with the combination of SNX-5422 and ibrutinib exhibit prolonged survival and decreased disease burden in the peripheral blood compared to the vehicle or single agent groups. In summary, we show that SNX-5422 degrades C481S BTK in vitro and prolongs survival when combined with ibrutinib in vivo. We therefore propose that upon molecular relapse, the use of SNX-5422 may prevent clinical relapse and deepen the response to ibrutinib. A phase 1 study is currently open at Ohio State to investigate whether the addition of SNX-5422 to an established dose of ibrutinib can eliminate the C481S mutant clone and prevent or delay disease progression in subjects with CLL (NCT02914327). EH and JAW contributed equally as senior authors. Citation Format: Timothy L. Chen, Bonnie Harrington, Jean Truxall, Ronni Wasmuth, Amy Lehman, Eric Orlemans, John C. Byrd, Jennifer A. Woyach, Erin Hertlein. Preclinical efficacy of the HSP90 inhibitor SNX-5422 in targeting C481S mutant BTK and ibrutinib resistant CLL [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1964.

Potent anti-leukemic activity of a specific cyclin-dependent kinase 9 inhibitor in mouse models of chronic lymphocytic leukemia.

Onset of progression even during therapy with novel drugs remains an issue in chronic lymphocytic leukemia (CLL). Thus, there is ongoing demand for novel agents. Approaches targeting cyclin-dependent kinases (CDK) have reached the clinical trial stage. CDK9 mediating RNA transcriptional elongation is the evolving pivotal CLL CDK inhibitor target. However, more CDK9 selective compounds are desirable. Here, we describe the CDK9 inhibitor LDC526 displaying a low nanomolar biochemical activity against CDK9 and an at least 50-fold selectivity against other CDKs. After demonstrating in vitro MEC-1 cell line and primary human CLL cell cytotoxicity we evaluated the LDC526 in vivo effect on human CLL cells transplanted into NOD/scid/γcnull (NSG) mice. LDC526 administration (75 mg/kg) for 5 days resulted in a 77% reduction of human CLL cells in NSG spleens compared to carrier control treatment. Next, we longitudinally studied the LDC526 impact on circulating CLL cells in the TCL1 transgenic mouse model. LDC526 (50 mg/kg) administration for two days led to a 16-fold reduction of blood CLL cell numbers. Remarkably, residual CLL cells exhibited significantly increased intracellular BCL-2 levels. However, the LDC526 cytotoxic effect was not restricted to CLL cells as also declining numbers of normal B and T lymphocytes were observed in LDC526 treated TCL1 mice. Taken together, our in vivo data provide a strong rational for continued LDC526 development in CLL therapy and argue for the combination with BCL-2 inhibitors.

Loss of NFAT2 Leads to an Acceleration of Clonal Evolution in CLL

Chronic lymphocytic leukemia (CLL) may be defined as a clonal expansion of mature B cells with stereotypic B cell receptors (BCR). Somatic hypermutation of the BCR heavy chains (IGVH) defines subgroups with different prognosis. In up to 10% of patients Richter's transformation to a high grade lymphoma with a mostly dismal prognosis is observed. The TCL1 transgenic mouse is a well accepted model of human CLL. Upon B cell-specific knock out of NFAT2, TCL1 mice develop a disease resembling Richter's syndrome with a significantly more aggressive disease phenotype. While TCL1 B cells exhibit tonic anergic BCR signaling characteristic of human CLL, loss of NFAT2 leads to readily activated BCRs indicating different BCR usage with altered downstream signaling.Here, we analyzed BCR usage in C57BL/6 wildtype, TCL1, and TCL1-NFAT2-/-mice. Splenocyte cDNA of the respective animals at an age of 7 months was amplified by multiplex PCR covering known heavy chain VDJ alleles. The PCR products were subsequently cloned into standard bacterial plasmids and subjected to conventional DNA sequencing. In addition, PCR products were analyzed by next-generation-sequencing (NGS). For data analysis, the IMGT/HightV-QUEST online tool was applied.A very diverse and polyclonal BCR usage was found in wild type mice with more than 4000 different clones identified. Although TCL1 mice at 7 months of age exhibit all features of CLL, their BCR usage as analyzed by unambiguously identified VDJ recombination was still polyclonal. Only with respect to VH usage, TCL1 mice were found to use a more limited set of V alleles compared to wild type mice. Loss of NFAT2 by conditional knockout on the contrary, lead to an oligoclonal usage of VDJ recombination, to a further limitation of V alleles, and also to the usage of identical VDJ recombination in different mice with high frequency - indicating BCR selection either by antigen or self-signaling in these mice. Further analyses by sequencing of hypervariable regions showed preferential usage of mutated BCRs in TCL1 mice and of unmutated BCRs in NFAT2 ko mice reflecting benign and aggressive disease in humans.In summary, the loss of NFAT2 signaling in CLL prefers the selection of unmutated BCRs as well as the preferential usage of certain VDJ recombinations which results in the accelerated development of true oligoclonal disease. DisclosuresNo relevant conflicts of interest to declare.

Exploring the Functional Relevance of BTK Beyond Chronic Lymphocytic Leukemia (CLL) Cells: BTK Expression in Non-Malignant Immune Cells of the Microenvironment Mediates CLL Development and Progression In Vivo

Background:Bruton's tyrosine kinase (BTK) is a critical component of the BCR pathway, but BTK and/or BTK-related enzymes such as ITK, Tec, BMX and RLK are also expressed in T, NK and myeloid cells, all of which are important sources of CLL-associated immune dysfunction. Whether BTK and/or BTK-related enzymes within non-malignant immune cells have any effects on CLL progression and immune function is not known. This is highly relevant translationally, as newer generations of BTK inhibitors selectively target BTK while sparing BTK-related enzymes in other cells. As the effects of constitutively active BCR and BTK targeting can be adequately modelled in the TCL-1 and TCL-1xXID mouse models (Woyach et al. Blood 2014), we used these preclinical tools to investigate if BTK expression in non-malignant immune cells modulates CLL progression in vivo. Our aims were: (1) to characterize differences in phenotype and function of T, NK and myeloid cells in leukemia-free and leukemic WT and BTK-inactive mice; (2) to assess BTK-related PD-L1 expression; and (3) to evaluate the in vivo relevance of BTK in non-malignant immune cells to CLL progression.Methods:Single-cell suspension cells were obtained from spleens from CLL-free matched WT B6 and BTK-inactive XID mice (n=11 each) and leukemic TCL-1xXID and TCL-1 mice (n=10 each). Flow cytometry was used to characterize the phenotype of B, T, NK and myeloid cells and myeloid-derived suppressor cells (MDSCs), PD-L1 expression, and T-cell degranulation. For survival experiments, WT B6 (n=29) and XID (n=27) mice were injected with 5x106 splenocytes from leukemic TCL1 donors and monitored daily. For in vivo depletion of monocytes/ macrophages, WT and XID (both n=12) mice received clodronate i.p. every 3 days from day 1 after injection of 3x107 splenocytes from leukemic TCL1 donors, and were euthanized after 36 days of treatment. RT qPCR was conducted on BTK-deficient XLA cell lines.Results:To explore whether BTK expression in non-malignant immune cells affects CLL development in vivo, we first conducted adoptive transfer experiments into WT and BTK-inactive XID mice. While WT mice had a median survival of 52 days after CLL development, survival was almost twice as long (90 days) in XID mice, indicating that BTK inactivation in non-leukemic cells prolongs survival. To identify whether this could be explained by differences in immune cell populations, we next characterized phenotype and function of T, NK and myeloid cells in leukemia-free and leukemic BTK functional and BTK-inactive mice. This comparison revealed that CLL-induced T-cell changes were broadly recapitulated in BTK-inactive mice, while significantly more immature (p=0.0206) and mature (p=0.008) NK cells were maintained. In the myeloid compartment, BTK activity did not affect the overall quantity of CD11b+ myeloid cells, but led to significant qualitative CLL-associated changes: while mice with functional BTK exhibited a significant CLL-driven loss of neutrophils (p=0.0002) and expansion of F4/80+CD11b+ macrophages (p=0.0001), leukemic BTK-inactive mice largely maintained these populations, suggesting anti-tumor properties of these cells. We next depleted myeloid cells with presumed BTK-associated anti-tumor properties in CLL-engrafted mice using clodronate. After 36 days of treatment, differences in spleen weights, CLL load in spleen and blood, and percentage of proliferating CLL cells were lost between WT and XID mice (all p>0.05), indicating that monocytes/ macrophages in XID mice indeed have in vivo anti-tumor functions. Neutrophils were expanded in both WT (median 44.7% of CD11b+ cells) and XID mice (43.7% vs. 6.1% in non-depleted mice). Interestingly, PD-L1 expression as a surrogate marker for tumor-associated immune suppression was generally significantly increased on antigen-presenting cells from BTK-inactive leukemic mice (CLL cells p=0.0002; inflammatory monocytes p=0.0014; patrolling monocytes p=0.001; MDSCs p=0.0201). RT qPCR experiments using XLA cell lines with modified BTK expression revealed that absence of BTK was associated with significantly higher PD-L1 RNA expression (p=0.0075), indicating a direct mechanistic link between BTK and PD-L1.Conclusions: Our data indicate that BTK in myeloid and NK cells directly mediates CLL development and progression in vivo. Targeting BTK in those cells in addition to CLL cells might provide substantial clinical benefits. DisclosuresWoyach:Acerta: Research Funding; Karyopharm: Research Funding; Morphosys: Research Funding.

BRAFV600E Accelerates Disease Progression and Increases Immune Suppression in a Mouse Model of B-Cell Leukemia

Background: Mitogen-activated protein kinase (MAPK) pathway components are frequently mutated in cancer, and abnormal MAPK signaling can drive malignancy by promoting tumor survival and proliferation. Furthermore, work in solid tumors shows that the presence of BRAFV600E further enhances tumor-induced immune suppression, but if and how this occurs in a disseminated disease such as leukemia is unknown. MAPK mutations occur in nearly 100% of classic hairy cell leukemia (HCL) and approximately 10% of chronic lymphocytic leukemia (CLL). Furthermore, up to 30% of B cell lymphomas exhibit activated MAPK signaling via expression of a BRAF pseudogene. However, the pathomechanisms of these abnormalities and the clinical utility of MAPK inhibitors in hematologic malignancies are unclear, and few models are available to evaluate this. To study these aspects, we developed in vitro and in vivo models of BRAF-mutated B cell malignancy.Methods: To mechanistically interrogate the role of BRAFV600E in malignant B cells, we transfected the CLL cell line OSUCLL with doxycycline-inducible constructs containing normal BRAF or BRAFV600E and incubated the cells -/+ doxycycline. These cells were also co-cultured with healthy donor T cells pre-incubated with anti-CD3/anti-CD28, and T cell proliferation was measured by flow cytometry. Cytokines and surface proteins were assessed by flow cytometry. To mimic human HCL, we developed a transgenic mouse model of BRAFV600E B cell leukemia. Mice with Cre-activatable BRAFV600E expression were crossed with mice carrying Cre under the control of the CD19 promoter. CD19-Cre x BRAFV600E or CD19-Cre only mice were then crossed with the well-characterized Eµ-TCL1 model of CLL to generate mice with spontaneous B cell leukemia expressing either wild-type BRAF (CD19-Cre x TCL1) or BRAFV600E (BRAFVE x CD19-Cre x TCL1) under the native BRAF promoter. B cell restricted expression of BRAFV600E was confirmed by immunoblots of purified B and T cells using a BRAFV600E specific antibody. Blood and spleen cells were examined by flow cytometry. For adoptive transfer experiments, leukemia cells (2x10^7) from transgenic mice were introduced intravenously into syngeneic healthy adult animals.Results: OSUCLL cells expressing BRAFV600E showed no significant changes in growth vs. wild-type BRAF expressing cells, but more strongly inhibited anti-CD3/CD28-induced proliferation of normal donor T cells. Transwell assays showed this effect was due both to soluble and contact-dependent factors. Expression of PD-L1 was not different between cells expressing normal and mutated BRAF, indicating this common checkpoint molecule is not the reason for greater immune suppression in this context. TNF levels were higher in BRAFV600E expressing cells and reduced by vemurafenib, but a TNF neutralizing antibody did not alter the inhibitory effect of BRAFV600E expressing cells on T cell proliferation. BRAFV600E x CD19-Cre x TCL1 mice developed B cell leukemia significantly earlier (median 4.9 vs. 8.1 months; P<0.001) and had significantly shorter lifespan (median 7.3 vs. 12.1 months; P<0.001) than their wild-type BRAF counterparts. In contrast to effects of BRAFV600E described in some solid tumors, BRAFV600E expression in B cells had no impact on the rate of B cell proliferation in vivo and only modestly reduced spontaneous apoptosis. To study the effects of this activating mutation on tumor-mediated immune suppression in vivo, leukemia cells from BRAFV600E x CD19-Cre x TCL1 or CD19-Cre x TCL1 mice were adoptively transferred into syngeneic mice. In 3 separate studies, tumor cells from BRAFV600E mice produced leukemia (>10% CD5+/CD19+ cells in blood) sooner than CD19-Cre x TCL1 tumor cells. In mice matched for disease load, BRAFV600E B cells produced a greater negative impact on T cells as evidenced by lower overall percentage of T cells, increased expression of T cell exhaustion markers PD-1, CD244, and CD160, and higher percentage of CD44+ memory T cells. Current studies are investigating the mechanism of these effects in vivo as well as the potential for pharmacologic reversal.Conclusions: Together, these results demonstrate the immune-suppressive impact of BRAFV600E in B-cell leukemias and introduce a novel mouse model to develop rational combination strategies to both directly target the tumor cell and overcome tumor-mediated immune evasion. DisclosuresLozanski:Genentech: Research Funding; Stemline Therapeutics Inc.: Research Funding; Beckman Coulter: Research Funding; Boehringer Ingelheim: Research Funding.

CD1d expression on chronic lymphocytic leukemia B cells affects disease progression and induces T cell skewing in CD8 positive and CD4CD8 double negative T cells.

Chronic lymphocytic leukemia develops within a complex network driven by genetic mutations and microenvironmental interactions. Among the latter a complex interplay with the immune system is established by the clone. Next to a proposed recruitment of support from T and myeloid cells, potential anti-CLL immune reactions need to be subverted.By using TCL1 mice as a CLL model, we show that TCR-Vβ7+ NK1.1+ T cells are overrepresented in this disease model and constitute a main subset of peripheral CD3+ cells with biased TCR usage, showing that these cells account for a major part for T cell skewing in TCL1 mice. Moreover, we show that overrepresentation is dependent on CD1d expression in TCL1 mice, implicating that these cells belong to a NKT-like cell fraction which are restricted to antigen presented by the MHC-like surface marker CD1d. Accordingly, we observed a high fraction of CD161+ cells within overrepresented T cells in CLL patients and we found downregulation of CD1d on the surface of CLL cells, both in TCL1 mice and patients. Finally, we show that in TCL1 mice, CD1d deficiency resulted in shortened overall survival. Our results point to an interaction between CLL and CD161+ T cells that may represent a novel therapeutic target for immune modulation.

The Eµ-Myc/TCL1 Transgenic Mouse As a New Aggressive B-Cell Malignancy Model Suitable for Preclinical Therapeutics Testing

Background: Aggressive B-cell lymphomas occurring in the setting of Chronic Lymphocytic Leukemia (CLL) as a large cell transformation are an important clinical problem, and improved mouse models to test novel and targeted therapeutics are needed. The Eµ-Myc mouse overexpresses c-Myc gene which is placed under control of the Myc promoter and lymphoid-specific IgH enhancer (Eµ), resulting in c-Myc overexpression and spontaneous B-cell lymphoma development. The Eµ-Myc mice have been used in drug development, however malignancy develops at variable ages and with differing genetics and response to therapeutic agents, making drug studies difficult. The Eµ-TCL1 transgenic mouse overexpresses the human TCL1 oncogene, under the control of the B-cell specific IgVH promoter and Eµ enhancer. Mice develop a spontaneous mature B-cell leukemia after a long latency period and represent a well-established model of human CLL. We crossed the Eµ-Myc and TCL1 mice to create a new model of aggressive B-cell lymphoma to test novel therapeutics that would be more homogeneous than the Eµ-Myc model.Methods: Eµ-Myc and TCL1 mice on C57BL/6 background strain were crossed and resulting genotypes were verified by PCR. All mice were kept in standard pathogen-free housing until death or removal from study. All mice born within a 24 month time period were followed for survival. At the time of death, spleen, lymph nodes, bone marrow, and liver were fixed in 10% neutral buffered formalin, embedded in paraffin, sectioned at 4uM, and stained with hematoxylin and eosin for histologic evaluation. For engraftment studies, adoptive cell transfer was done by injecting fresh splenic white blood cells (1e7 cells, >75% CD5+/CD19+) into the tail vein of C57BL/6 wild type recipient mice. In the drug treatment experiments ibrutinib (~30mg/kg/day) was administered in drinking water and KPT-8602 (15mg/kg) by daily oral gavage. Survival was shown using Kaplan-Meier curves and survival curves compared statistically using a log-rank test.Results: The Eµ-Myc/TCL1 mice had significantly worse survival (median 45.5, range 29-74 days) compared to Eµ-Myc (median 118, range 42-520 days) or TCL1 (median 359.5, range 188-531 days) mice (p=<0.0001). Survival curves are show in Figure 1. Pathologic examination revealed aggressive B-cell histology similar to Burkitt lymphoma in both the Eµ-Myc mice and Eµ-Myc/TCL1 mice, whereas the TCL1 mice showed mature B-cell morphology. Infiltration of the bone marrow of Eµ-Myc and Eµ-Myc/TCL1 mice was extensive in comparison with TCL1 mice. In all three mouse genotypes the normal architecture in the spleen was effaced by neoplastic cells. Adoptive transfer of Eµ-Myc/TCL1 splenic lymphocytes produced similar pathologic findings to the Eµ-Myc/TCL1 transgenic mice with less severe splenic enlargement in the engrafted mice. Median survival of the engrafted mice was short (38 days). There was a differential response to targeted therapeutics between the groups. Ibrutinib, an irreversible inhibitor of Bruton tyrosine kinase (BTK) previously demonstrated to improve survival in TCL1 mice, had no survival advantage over vehicle treatment in either Eµ-Myc or Eµ-Myc/TCL1 mice (p=0.876 and P=0.83 respectively). KPT-8602, a second generation selective inhibitor of exportin 1 (XPO1) was tested in Eµ-Myc/TCL1 engrafted mice, and demonstrated a significant improvement in survival compared to vehicle alone (p=<0.0001). A first generation agent (selinexor) targeting XPO1 is currently in human clinical trials for aggressive lymphomas, including large cell transformation of CLL.Conclusions: Mice with both c-Myc and TCL1 transgenes develop an aggressive B-cell malignancy and have decreased survival compared to Eµ-Myc or TCL1 mice. The histology of the disease is similar to that of Eµ-Myc mice, but with shorter survival with less variability. The resulting mouse model has differential responses to targeted therapeutics, with a poor response to ibrutinib more similar to aggressive lymphoma than CLL. The malignant B cells can be adoptively transferred into immunocompetent mice for experimental drug treatment studies. These qualities make Eµ-Myc/TCL1 mice a useful tool to test new therapies for aggressive lymphoma where there is an unmet clinical need. Work is currently underway to further define the genetic characteristics of this mouse in comparison to human lymphoma sub-types and responsiveness to targeted agents. [Display omitted] DisclosuresByrd:Acerta Pharma BV: Research Funding.

CLL Disease Severity Is Enhanced in Tcl1 Mice Deficient for Pro-Apoptotic Regulator Noxa

Abstract Despite the development of new treatment strategies, chronic lymphocytic leukemia (CLL) remains an incurable disease. Apoptosis dysregulation caused by environmental signaling, predominantly within lymph nodes, plays a key role in CLL maintenance and treatment resistance. In earlier work, we demonstrated that expression of the pro-apoptotic Bcl-2 family memberNoxa is increased in circulating CLL cells compared to normal B-cells (1). Moreover, Noxa levels are decreased in CLL cells that reside in the lymph node while its key anti-apoptotic binding partner, Mcl-1, shows a reverse trend (2). This suggests that the decreased Noxa/MCL-1 ratio within the microenvironment may contribute to enhanced CLL survival. Here we aimed at elucidating a functional role for Noxa in CLL in the context of a murine model by crossing Noxa deficient mice (Noxa-KO) with Eμ.Tcl1-Tg mice (TCL1) mice. We first established that also in Tcl1 mice the Noxa/Mcl-1 ratio was increased PB vs. LN cells. The phenotype and distribution of leukemic cells was similar in Tcl1 and Noxa-KO/tcl1 mice. Interestingly, the accumulation of CLL cells in the peripheral blood was accelerated in Noxa-KO/TCL1 mice. This was accompanied by a reduction of apoptotic cells in the spleen of Noxa-KO/TCL1 mice. In accordance with these findings, survival of Noxa-KO/Tcl1 mice was decreased compared to the Tcl1 mice (342 days vs. 396 days, p&lt;0.001, Figure 1). Our data suggest a role for Noxa in CLL as a tumor suppressor, and provides a rationale for the use of compounds which may alter the Noxa/Mcl1 balance in patients. 1. W. J. Mackus et al., Chronic lymphocytic leukemia cells display p53-dependent drug-induced Puma upregulation. Leukemia19, 427-434 (2005). 2. L. A. Smit et al., Differential Noxa/Mcl-1 balance in peripheral versus lymph node chronic lymphocytic leukemia cells correlates with survival capacity. Blood109, 1660-1668 (2007). Figure 1. Survival analysis of Noxa-KO/TCL1 mice and TCL1 mice. The Kaplan-Meier curves were analyzed by a Log-Rank test. Figure 1. Survival analysis of Noxa-KO/TCL1 mice and TCL1 mice. The Kaplan-Meier curves were analyzed by a Log-Rank test. Disclosures No relevant conflicts of interest to declare.

Genetic Inhibition of PI3K p110delta Antagonizes Survival Signals and Induces Immune Activation in Chronic Lymphocytic Leukemia (CLL)

Background: Chronic lymphocytic leukemia (CLL), the most common adult leukemia in the western world, is characterized by the accumulation of malignant mature B cells in the blood, lymph nodes, spleen and bone marrow. CLL cells display up-regulated B cell receptor (BCR) activation, which maintains B cell survival and proliferation through transmitting microenvironmental stimuli. Due to aberrant regulation of the BCR, CLL cells display constitutively activated survival and proliferation pathways, such as phosphoinositide-3 kinase (PI3K) p110δ pathway. In addition, immune tolerance, a critical mechanism for cancer immune invasion, remains a major barrier for effective anti-cancer therapy. PI3K p110δ also plays critical roles in T cell development and function. Studies using genetic p110δ kinase-dead mice have showed that p110δ inhibition can reverse the tumor induced immune tolerance, impairs the expansion of regulatory T cells (Treg) thereby enhancing the cytotoxic CD8+ T cells to induce tumor regression in various solid tumor models.Methods: Here we use a PI3K p110δ kinase-dead (δD910A/D910A) mouse model and a well-characterized Eμ-TCL1 mouse CLL model to delineate the role of p110δ signaling in CLL leukemia pathogenesis and microenvironmental immune tolerance. The δD910A/D910A has a point mutation on the p110δ kinase domain, which results in the complete inactivation of p110δ activity without affecting the expression of p110δ or the other isoforms. TCL1 mice exhibit B-cell leukemia similar to that in humans beginning around 4-6 months. Adoptive transfer experiments are performed by tail-vein injection of 5e6 splenic lymphocytes from a leukemic Eμ-TCL1 mouse. Leukemia progression is measure by flow cytometry for the CD5+ CD19+ % lymphocytes in the peripheral blood. Whole blood cytotoxicity assay was performed by incubating whole blood from recipient mice with cryopreserved cells from the original donor to assess the their cytotoxic effects towards the donor cells.Results: δD910A/D910A mice were crossed with TCL1 mice to examine the effect of systemic p110δ inactivation in CLL. We show that global inactivation of PI3K p110δ significantly prevented spontaneous leukemia development. Both δWT /TCL1 and δWT/D910A /TCL1 mice developed CLL like disorder starting 4 months whereas δD910A/D910A /TCL1 mice did not show any sign of disease up to 14 months. This indicates that p110δ is a critical kinase for CLL disease initiation and expansion.We then performed adoptive transfer of TCL1 leukemic cells into p110δ mice to examine the role of p110δ in the microenvironment. We show that inhibiting p110δ in the microenvironment impairs leukemia engraftment. On day 31 after engraftment, 7 out of 10 mice from the δWT group developed leukemia while no mice from δD910A/D910A group had detectable leukemia. On day 43, δD910A/D910A group started to have leukemia incidence (2 out of 7) when δWT group continued to progress. δWT/D910A group showed intermediate leukemia incidence and disease development, which were different from the phenotype we saw in δWT/D910A /TCL1. This suggests that p110δ plays a different role outside of the leukemia compartment.To identify potential effector cells, we performed adoptive transfer of ovalbumin (OVA) -expressing TCL1 leukemia cells into p110δ mice. The TCR (OT1) specific SIINFEKL peptide of OVA is used to induce the expansion of OVA antigen specific CD8+ T cells. Interestingly, upon engraftment, δWT and δWT/D910A mice showed similar levels (4% and 3.5%, respectively) of OVA specific CD8+ T cells whereas the δD910A/D910A mice only have 2%. This appears to be contradicting to the less leukemia development we observed previously. However, in vitro whole blood killing assay showed that whole blood from δD910A/D910A mice exhibited higher cytotoxicity to TCL1 leukemia cells despite their impaired development of the antigen specific anti-tumor CD8+ T cells.Conclusions: This study demonstrates that blocking p110δ in CLL not only abrogates survival signals in leukemic cells but also has the potential to reverse immune suppression in the micoenvironment. Studies are ongoing to pinpoint the effector cell subsets. The finding from this study will suggest that idelalisib, the PI3K p110δ inhibitor recently approved by FDA for the treatment of CLL, or other inhibitors of this pathway can potentially be used to treat a wide range of cancers by unleashing host anti-tumor immunity. DisclosuresByrd:Acerta Pharma BV: Research Funding.

Overexpression of Activation-Induced Deaminase in TCL1 Mice Leads to the Development of IGHV -Mutated and -Unmutated CLL Clones That Resemble Unique Subsets of Human CLL

Introduction. Somatic hypermutation (SHM) and class-switch recombination (CSR) are critical physiologic events in an effective normal B-cell immune response. Notably, both SHM and CSR are initiated by activation-induced cytidine deaminase (AID). In CLL, IGHV clonal mutations correlate strongly with better clinical outcomes. Eμ-T-cell leukemia-1 (TCL1) transgenic (Tg) mice are a valuable model of CLL. However because SHM and CSR occur rarely in the leukemic clones from these animals, TCL1 Tg mice mimic only IGHV -unmutated CLL and hence may not provide an understanding of the roles of SHM and CSR in disease evolution. To address these issues, we developed two new TCL1 strains by interbreeding mice over-expressing AID in all cells (Em-TCL1xActin-AID) or only in B lymphocytes (Em-TCL1xVκ-AID).Methods. B-cell clonal expansions were identified in spleen cells from 22 TCL1 and 33 TCL1xAID Tg (10 Em-TCL1xActin-AID plus 23 Em-TCL1xVκ-AID) mice at 10-20 months of age. This was done by amplifying cDNAs by PCR using consensus IGHV FR and IgM-, IgG-, and IgA-specific primers for IgH chains and Vκ FR and Cκ for IgL chain genes. DNA sequences of these amplicons were compared to murine germline IGHV s and IGKV s, and IGHV-D-JH and IGKV-JK rearrangements by IMGT V-Quest. Because there were no major differences in the parameters listed below for the two TCL1xAID Tg mouse strains, data were combined.Results.Clonal expansions: Monoclonal/oligoclonal expansions were detected in all TCL1 mice; these used only µ H and κ L chains. Similar expansions were detected in 31 of 33 TCL1xAID mice; each animal bore an IgMκ+ clone and 7 also exhibited an IgGκ+ clone.IGHV gene use in IgMκ+ clones: IGHV use did not differ significantly between IgMκ+ TCL1 and IgMκ+ TCL1xAID clones. Approximately 50% used VH1-55, VH11-2, or VH12-3, some of which encoded stereotyped anti-phosphatidylcholine antibodies.SHM: Among the IgM+ TCL1 clones, there was a mutation frequency of 0.05% for IGHV and 0.09% IGKV. In contrast, the IGHV and IGKV mutation frequencies were considerably higher in TCL1xAID mice. Specifically, for IGHV, the TCL1xAID animals displayed a frequency of 0.47% in IgM+ and 3.0% in IgG+ clones; for IGKV, the frequency was 0.9% for IgM+ and IgG+ combined. Consistent with these mutations being mediated by AID, mutations localized more frequently in AID hotspots than coldspots at a ratio of ≥7:1.However, SHM did not affect all clones equally. Although the mutation frequency in VH12-3 and VH11-2 clones was higher (0.38%, range: 0-1.9%) than the TCL1 level (0.05%), it was considerably less than that found in the TCL1xAID clones using other IGHV s (0.80%, range: 0-3.8%). In addition, no mutations were detected in VH1-55 clones. Consistent with a reduced AID mutational activity in clones expressing these specific genes, none of the genes were found in the isotype-switched, IgG-expressing clones.IGHV gene use and SHM in clones that underwent CSR: Notably, in only 2 of 9 instances was the same IGHV-D-J rearrangement found in IgMκ+ and IgGκ+ clones from the same mice; these IgM+ and IgG+ shared clones used VH5 genes. For the remaining 7, only the IgG+ version was detected; all but one of these used a VH1 gene. Also, within the IgG-only group, IGHV1-47 was used by 2 different clones and these were highly mutated (8.9%).Stereotyped IGHV-D-J and IGκ V-Jκ rearrangements: Among 43 IgM clones from TCL1xAID mice, we found 8 clones expressing VH11-2 and Vκ14-126 and 10 clones expressing VH12-3 and Vκ4-91; these IGHV-D-J and IGκV-Jκ rearrangements were very similar to canonical anti-phosphatidylcholine-producing clones.Summary and conclusions.Over-expression of AID in TCL1 mice leads to markedly increased SHM and CSR. However, SHM is not equivalent for all IGHV genes since despite AID over-expression certain IGHVs and IGKVs appeared less sensitive to major increases in the extent of SHM and the occurrence of CSR. This property resembles some human CLL IGHV s that rarely develop SHMs or undergo CSR despite the B-cell's ability to synthesize AID (e.g., many IGHV1-69+ clones). AID overexpression also led to IgG+ clones for which an IgM precursor was not found. This resembles those human stereotyped CLL clones that are only found as IgGs (e.g., stereotyped subsets 4 and 8). Finally, the two new TCL1xAID mouse strains described provide new models to study IGHV -mutated and IGHV -unmutated CLL and represent novel tools to evaluate the role of AID in leukemic progression. DisclosuresNo relevant conflicts of interest to declare.

The Role of Tetraspanin CD37 in B-Cell Malignancy

Members of the tetraspanin superfamily of transmembrane proteins have roles in a variety of processes including cellular adhesion, migration, activation, proliferation, survival, and solid tumor metastasis. Although numerous tetraspanin proteins are expressed by immune cells, only a few have expression that is restricted to these cells. CD37 is not found in non-hematologic tissues and is highly expressed by mature B-cells, with greater than 15 times higher expression compared to other immune cells (Deckert et al., 2013). Recently there has been significant interest in CD37 as a therapeutic target in non-Hodgkin lymphoma and Chronic Lymphocytic Leukemia (CLL), as evidenced by the ongoing clinical development of four different anti-CD37 therapies. However, very little is known about the biological function of this protein. Ligation by the CD37-targeting peptide otlertuzumab leads to induction of both pro-survival Akt activation and pro-apoptotic SHP1 signaling, although cellular death is heavily favored (Lapalombella et al., 2012). Furthermore, CD37 is critical to normal plasma cell development, clustering VLA-4 (α4β1 integrin) on the surface of germinal center B-cells to allow integrin-mediated activation of Akt (van Spriel et al., 2012). In CLL, expression of the VLA-4 subunit CD49d is associated with more aggressive disease and promotes homing and adhesion to supportive cells within lymphoid tissues. This prompted us to explore the role of CD37 expression in B-cell malignancy using a CD37-deficient Eµ-TCL1 mouse model of CLL. For these studies, we monitored mice that were CD37 wild type, heterozygous knockout, or homozygous knockout (all hemizygous for the TCL1 transgene). While no significant difference was observed in the time until appearance of leukemia in peripheral blood, loss of CD37 led to decreased survival of TCL1 mice (p=0.032). In the same timeframe, non-TCL1 mice with CD37 deficiency did not exhibit impaired survival. While CD37 is predominantly seen in B-cells, the protein serves a role in other immune cells despite substantially lower expression. Our results are consistent with the recent reports on impaired dendritic cell migration in CD37 deficient mice, which is associated with decreased anti-tumor immunity in the context of a subcutaneously injected mouse cell line expressing a foreign human antigen (Gartlan et al., 2013). Our data support a potential role for CD37 in anti-tumor immune response against the syngeneic leukemia that arises spontaneously in the TCL1 model of CLL. Given the complex nature of this global knockout model, other functions of CD37 may be obscured. Ongoing studies will examine engraftment of CD37-deficient or wildtype TCL1 leukemia into healthy mice to elucidate whether the B-cell intrinsic role of CD37 is supportive of the leukemia, independent of an extrinsic role in anti-tumor immunity. [Display omitted] DisclosuresByrd:Acerta Pharma BV: Research Funding.

NFAT2 Regulates Anergy Induction in CLL through Lck

NFAT is a family of highly phosphorylated proteins residing in the cytoplasm of resting cells. Upon dephosphorylation by calcineurin, NFAT proteins translocate to the nucleus where they orchestrate developmental and activation programs in diverse cell types. CLL is a clonal disorder of mature B cells characterized by the expression of CD19, CD23 and CD5. With respect to prognosis, it constitutes a heterogeneous disease with some patients exhibiting an indolent course for many years and others progressing rapidly and requiring early treatment. A defined subgroup of patients shows enhanced responsiveness to stimulation of the B cell receptor (BCR) complex and more aggressive disease. In contrast, another subset of CLL patients with more indolent course is characterized by an anergic B cell phenotype referring to B cell unresponsiveness to IgM ligation and essential lack of phosphotyrosine induction and calcium flux. Here, we analyzed the role of NFAT2 in the pathogenesis of B-CLL and in anergy induction in CLL cells.For this purpose, we generated conditional CD19-Cre NFAT2 knock out mice, which exhibit NFAT2 deletion limited to the B cell lineage. To investigate the role of NFAT2 in the pathogenesis of CLL, we used the Eµ-TCL1 transgenic mouse model. TCL1 transgenic mice develop a human-like CLL at the age of approximately 14 weeks to which the animals eventually succumb at an average age of 10 months. We generated TCL1+NFAT2 ko mice with TCL1 transgenic mice without an NFAT2 deletion serving as controls. To identify novel NFAT2 target genes in CLL cells, we also performed a comparative gene expression analysis on CLL cells with intact NFAT2 expression and on CLL cells with NFAT2 deletion using affymetrix microarrays. In order to asses the anergic phenotype in CLL cells and the role of NFAT2 in its induction, we performed Ca2+ mobilization assays using a flow cytometric approach and performed Western Blots for multiple downstream signaling molecules.Mice with NFAT2 ko exhibited a significantly more aggressive disease course with accelerated accumulation of CD5+CD19+ CLL cells in different organs, significantly higher proliferation rates and a dramatically reduced life expectancy (200 vs. 325 days) as compared to TCL1 control animals. To identify NFAT2 target genes responsible for the observed alterations in the disease phenotype, we subsequently performed a gene expression analysis with CLL cells from both leukemic cohorts. Here, we detected a substantially altered expression profile of genes associated with B cell anergy in the TCL1+NFAT2 ko mice. The vast majority of these genes was expressed significantly less in the absence of NFAT2 with Lck, Pacsin1 and the E3 ligase Cbl representing the biggest hits.To further delineate the anergic phenotype and the role of NFAT2 in its induction, we subsequently performed Ca2+ mobilization assays. While anergic CLL cells from TCL1 mice exhibited an unresponsive phenotype with respect to Ca2+ flux upon IgM ligation, TCL1+NFAT2 ko mice showed an entirely normal capacity to mobilize intracellular Ca2+. Furthermore, IgM stimulation did not activate normal phosphotyrosine induction (phosphorylation of AKT and ERK kinases) in TCL1 mice while NAFT2-deficient CLL cells exhibited an unremarkable activation pattern with respect to AKT and ERK as assessed by Western Blotting. NFAT2-deficient CLL cells on the contrary exhibited compromised activation of the anergy regulator Lck as assessed by Y394 phosphorylation. Bypassing the BCR by antigen-independent stimulation with CD40 and LPS demonstrated slightly increased proliferation in anergic TCL1 CLL cells while NFAT2-deficient CLL cells exhibited massive proliferation.In summary, our data provide strong evidence that genetic loss of NFAT2 leads to more aggressive disease in CLL which is associated with the loss of the anergic phenotype. We could show that NFAT2 controls the expression of several important anergy-associated genes and identified Lck as a critical target of NFAT2 in this context. Taken together, our data demonstrate that the NFAT2-Lck axis plays an essential role in the pathogenesis of CLL and implicate it as a potential target in its treatment. DisclosuresNo relevant conflicts of interest to declare.

Targeting Dysfunctional Myeloid Cells Delays Disease Development and Improves Immune Function in a CLL Mouse Model

Chronic lymphocytic leukemia (CLL) is a malignancy of mature B cells that is characterized by apoptosis resistance and dysfunctional immune system. The chronic nature and slow development of the disease indicates a contribution of CLL-induced inflammation in the disease course. Previous reports suggested a potential role of myeloid cells in mediating these defects. However, the composition and function of CLL-associated myeloid cells have not been thoroughly investigated in an in vivo system. Here, we used the well-established CLL mouse model, Eµ-TCL1 mice (TCL1), to characterize changes within myeloid cell populations along with CLL development and the influence of their depletion on disease progression and immune dysfunction.We have recently shown that CLL development in TCL1 mice is associated with massive changes within myeloid cell populations. In the peritoneal cavity (PC) of leukemic mice we observed an infiltration of monocytes and an M2-like skewing of macrophages according to phenotypical and signaling signatures. Along this line, monocytes infiltrated the spleens of leukemic animals, both in primary CLL and adoptive transfer models, which is most likely due to high CCL2 serum levels. These monocytes lost the inflammatory Ly6Chi subset and were severely skewed towards Ly6Clow patrolling monocytes, accompanied by high expression of adhesion and angiogenic molecules like ICAM1, PECAM1 and MMP14. Gene expression profiling of splenic myeloid cells from TCL1 mice revealed an enrichment of various genes involved in dendritic cell (DC) maturation and MHC-II-mediated antigen presentation. However, the numbers of MHC-IIhi mature DCs and macrophages were significantly decreased, suggesting a monocyte differentiation arrest leading to impaired anti-tumor immune response. The observed transcriptional upregulation of multiple inflammatory cytokines like TNF-α, CXCL9, CXCL10 and CXCL16 in monocytes was confirmed by serum cytokine arrays, and is likely due to the overexpression of the pro-inflammatory regulator TREM-1. In addition, TCL1 monocytes upregulated the expression of several inhibitory molecules like PD-L1, IL-10, IL1ra and IL4i1 suggesting an impaired immune function.While CLL-induced immune dysfunction is a well-established phenomenon, the contribution of myeloid cells in this context was not clear. We therefore sought to determine the in vivo effects of myeloid cell depletion on CLL development and its associated immune defects. For that purpose we used liposomal clodronate to selectively ablate macrophages and monocytes from young wild-type mice adoptively transferred with murine CLL. Our data clearly show control of CLL development in clodronate-treated mice relative to control liposomes as demonstrated by decreased spleen weight (1.09 vs. 0.54 g, p < 0.0001) and a significant drop in tumor load, defined as CD5+CD19+ cells, in spleen (60.58% vs. 42.25%), peripheral blood (43% vs 11.8%), PC (66.2% vs 3.1%), lymph nodes (4.9% vs 1.2%) and bone marrow (1.9% vs 0.8%). In addition, we observed changes in immune effector cells in response to myeloid cell depletion suggesting better immune status in treated mice. Interestingly, the loss of macrophages/monocytes was compensated by increased splenic monocyte proliferation as shown by EdU incorporation in vivo. In contrast to control mice, the repopulating monocytes upon clodronate treatment were largely inflammatory Ly6Chi monocytes.In summary, our data show that skewing of myeloid cells actively contributes to CLL development via; 1) enhancing the survival of leukemic cells, and 2) suppressing anti-tumor immune functions. In the absence of monocytes and macrophages, disease development is delayed in mice adoptively transferred with murine CLL. Therefore, we suggest that targeting non-malignant myeloid cells in CLL might serve as a novel strategy for CLL immunotherapy. DisclosuresNo relevant conflicts of interest to declare.

The Flavonoid Wogonin Reduces CLL Cell Survival in Vitro and Leukemia Development in Eµ-TCL1 Mice By Targeting Aberrant TNF Receptor Signaling

The importance of the tumor microenvironment in the development of B-cell chronic lymphocytic lekuemia (CLL) is now widely accepted. Previous studies within our and other groups revealed the establishment of an inflammatory milieu in CLL characterized by enhanced expression and secretion of several cytokines and their receptors. Using 250 CLL serum samples and 50 age-matched controls, we found significantly increased levels of the TNFα receptors TNFR1 and TNFR2 in the patients that positively correlated with an adverse clinical outcome. Based on these findings we aimed to investigate the functional role of TNFR signaling in CLL development.In contrast to healthy B cells that do not express TNFα receptors, we detected TNFR1 expression in CLL cells upon survival-inducing culture conditions and in lymph node sections of 80 CLL patients, where TNFR1 signals co-localized to the B cell marker CD20 and were mainly present within proliferation centers. These findings were confirmed in Eµ-TCL1 mice, a well-established mouse model for CLL. Here, CLL cells in the peripheral blood were negative for TNFR1. However, the cells upregulated the receptor upon entering the spleen where they get into contact with accessory cells, receive survival stimuli and undergo proliferation. In addition, increased levels of soluble TNFR1 in the serum were confirmed in the mice.After ligand binding, TNFR1 can activate two different signaling pathways: (1) the extrinsic apoptosis cascade through its death receptor domain, or (2) survival-inducing NFκB signaling. The latter pathway can be blocked by wogonin, a naturally occurring monoflavonoid. In a multitude of in vitro and in vivo studies, wogonin has been shown to exert anti-oxidant, anti-inflammatory and anti-tumor activities. By ex vivo treatment of CLL cells with TNFα we observed NFκB activation which was reversed by TNFα-blocking antibody, suggesting TNFR1-mediated survival signaling in CLL. Therefore, we aimed to test whether wogonin can prevent this signaling in CLL cells and might therefore represent a novel potential drug for CLL.In CLL cocultures, wogonin treatment resulted in a concentration-dependent apoptosis induction, which was significantly increased by the addition of TNFα. To test the effects of wogonin in vivo, we transplanted isogenic, immunocompetent wild-type mice with CLL cells from leukemic TCL1 animals and treated them daily with 40 mg/kg wogonin. When treatment was started 2 days after transplantation, wogonin significantly reduced spleen weights and lead to a reduced CLL content in the spleen, the bone marrow and the peritoneal cavity. If treatment was started in advanced disease stage, wogonin slightly lowered spleen weight and the CLL content in the spleen, whereas the percentage of CLL cells in the peripheral blood was increased. Interestingly, here wogonin treatment resulted in a loss of cell surface TNFR1 expression in splenic CLL cells and increased TNFR1 levels in the serum. These data suggest that wogonin induces a redistribution of CLL cells in vivo, preventing their homing to lymphoid organs and that loss of TNFR1 expression might be involved in this process.In summary, our results show that TNFR1 signaling is involved in CLL cell activation and survival. Targeting this pathway with wogonin reduces CLL cell viability in vitro and leukemia development in TCL1 mice. DisclosuresNo relevant conflicts of interest to declare.

Aberrant PD-L1 Expression in CLL As a Result of Adaptive Immune Resistance Mediated By Tumor-Secreted Circulating miRNA Binding to Toll-like Receptor 7

Background: Chronic lymphocytic leukaemia (CLL) is a model cancer to study immune evasion via PD-L1/PD-1 signalling: aberrantly expressed PD-L1 on CLL and PD-1 on CD8 T cells are key mediators of poor anti-tumor immune responses, which are a major hallmark of CLL. Several preclinical studies suggest that aberrant PD-L1 expression is a result of adaptive immune resistance and is induced during immune responses within the tumor microenvironment. It has recently been proposed that specific circulating microRNAs (miRNAs) shed by malignant cells participate in the complex crosstalk between cancer cells and microenvironment, and that they activate immune cells via Toll-Like Receptor (TLR) 7 due to their structural similarity to its natural ligand. In addition, TLR signaling has been demonstrated to result in the upregulation of costimulatory molecules such as CD80, CD86 and PD-L1/2. In CLL, aberrant circulating miRNA and TLR expression patterns have been well characterized. Therefore, we hypothesized that aberrant PD-L1 expression in CLL is a result of continuous TLR7 signaling mediated by circulating miRNAs. Our aims were to demonstrate that (1) specific circulating miRNAs induce PD-L1 expression and have functional consequences, (2) miRNA/PD-L1 associations are mediated via TLR7, and (3) miR/TLR/PD-L1 interactions are subject to the dynamics of CLL development.Methods and Materials: Mononuclear cells from spleen cell suspensions from 3 month old TCL1 transgenic, wild-type (WT) or TLR7-/- mice (total n=13) were treated ex vivo for 18hours with liposomal formulations of synthetic scrambled miRNA or miRNAs -16 (negative control), -21, -29, -150 and -155, which are reported to have an effect on immune cells and to be released by CLL cells. Specific TLR7 and TLR9 agonists were included for comparison. Primary human CLL cells and healthy B cells were treated with specific TLR2/6, TLR7 and TLR9 agonists. In adoptive transfer experiments, young WT mice (n=15) were injected with 4x107CLL cells from TCL1 transgenic mice. Mice were sacrificed at days 3, 6, 9, 12 and 15, and spleen cells were treated ex vivo as above. Changes in surface PD-L1, CD69 and CD86 expression on DAPI-negative CD19+ B cells/CLL cells were determined by flow cytometry. Supernatant cytokines were screened by multiplex ELISA.Results: PD-L1 surface expression on spleen B cells from both WT and non-leukemic TCL1 mice was strongly induced by miRs -21 and -29, and moderately by -150 and -155, but not by miR-16 negative control. The degree of PD-L1 upregulation by miRs -21 and -29 was comparable to the effect of direct TLR7 and TLR9 binding by specific agonists. Similar patterns were seen for CD69 and CD86 expression. Across treatment conditions, PD-L1 expression was highly correlated with the expression of CD69 (r .7777, p<.0001) and CD86 (r .7516, p<.0001). This observation strongly suggests that PD-L1 expression after TLR engagement is a marker of activation/costimulation, and therefore a physiological adaptive immune response to TLR binding in healthy B cells. Functionally, miR treatments resulted in increased IL-6, IL-10 and TNFα, with miR-29 having the strongest effect. PD-L1, CD69 and CD86 could also be induced by TLR engagement in healthy B cells, but not in CLL patient cells, where PD-L1 was confirmed to be already aberrantly expressed. To elucidate when in the course of CLL development PD-L1 expression becomes aberrant and if it ceases to be inducible by miR treatment, we sacrificed adoptively transferred mice every 3 days to simulate tumor development. With increasing CLL the magnitude of the fold-change of PD-L1 expression following miR treatment decreased substantially, and the baseline expression of miR-untreated B cells increased consistently until day 15. Interestingly, although the PD-L1 response was substantially decreased with tumor load, it was not completely abrogated, even on day 15 when mice had a median CLL load of 71%. Importantly, miR treatment did not result in increased PD-L1, CD69 or CD86 expression in B cells from TLR7-/- mice, indicating that the miR/PD-L1 interactions are indeed mediated by TLR7.Conclusions: Our findings support that PD-L1 expression on B cells can be induced by specific miRNAs known to be produced by CLL cells, and that this effect is mediated via TLR7. Therefore, aberrant PD-L1 expression on CLL is likely to be a result of adaptive immune resistance mediated by tumor cell-produced circulating miRNAs. DisclosuresGribben:Celgene: Research Funding; Pharmacyclics: Honoraria; Roche: Honoraria.

Proteinkinase C-β Inhibitors Mitigate Microenvironment-Mediated Survival Of CLL Cells and Sensitise Malignant B Cells To Cytotoxic Drugs

Heterotypic interactions between bone marrow stromal cells (BMSCs) and malignant B cells contribute to apoptosis-resistance of tumour cells, based on the provision of anti-apoptotic factors by stromal cells. For this reason, interference with the microenvironment may offer an alternative approach to chemotherapy to target B cell lymphomas/ leukaemias. However, the success of such treatments critically relies on specific targets expressed in the lymphoma microenvironment. We have previously reported that monoclonal B cells from patients with CLL, MCL und ALL activate and reprogram BMSCs. This activation of stromal cells is an essential prerequisite for microenvironment-mediated survival of malignant B cells and requires the induction and activation of protein-kinase C-β in stromal cells in vitro and in vivo. This is underscored by a complete resistance of PKC-β knockout mice to adoptively transferred B cell lymphomas from TCL1 transgenic mice (Lutzny et al. Cancer Cell. 2013 Jan 14; 23(1):77-92.).Analyses of primary CLL cells co-cultured on human or mouse BMSCs indicate that stromal cells protect malignant B cells from apoptosis primarily by enhancing the expression of the anti-apoptotic proteins Mcl1, XIAP, BclXL and Bcl2A1. Knockdown of Mcl1 expressed in CLL cells further demonstrates that its expression is crucial for stroma-mediated survival even in the presence of BMSCs.Since BMSC-mediated survival and propagation of CLL depends on the kinase activity of PKC-β, we hypothesized that pharmacological inhibition of the activation of stromal cells using small molecule inhibitors against PKC-β may display anti-leukemic effects. Here we report that the PKC-β inhibitor enzastaurin enhances the cytotoxic effects of standard chemotherapeutic drugs and of the BCL2-inhibitor ABT737. Dose-response analyses indicate that enzastaurin potentiates the cytotoxic effects of ABT737 in a synergistic manner, rapidly causing caspase-mediated apoptosis of malignant B cells. This drug-sensitising effect of enzastaurin is mediated by inhibition of PKC-β induced and expressed in activated stromal cells and not related to a direct cytotoxic effect on malignant B cells: PKC-β deficient BMSCs fail to maintain high expression levels of the anti-apoptotic proteins Mcl1, XIAP and Bcl2A1 in CLL cells, which show a significantly enhanced sensitivity to ABT737 compared to CLL cells cultured on PKC-β proficient stromal cells.These data provide evidence that PKC-β inhibitors can therapeutically be used to abolish microenvironment-mediated survival of malignant B cells. In order to assess whether drug combinations of enzastaurin and BCL2 inhibitors demonstrate synergistic anti-lymphoma effects in vivo, we adoptively transferred malignant B cells from diseased TCL1 mice into wild-type recipient mice. Tumour-bearing mice were treated with enzastaurin, ABT199 or a combination of both drugs. Response to treatment, assessed by PET-CT scan, indicates that the combination of enzastaurin and ABT199 is superior to single agent treatment.These data indicate that malignant B cells can be sensitized to cytotoxic drugs by inhibiting the tumour-promoting effects of the lymphoma microenvironment with PKC-β inhibitors. Our preclinical in vitro and in vivo experiments justify testing this drug combination as a new approach to leukaemia/ lymphoma therapy in a phase I/II clinical trial. Disclosures:No relevant conflicts of interest to declare.

A Sleeping Beauty screen reveals NF-kB activation in CLL mouse model

AbstractTCL1 oncogene is overexpressed in aggressive form of human chronic lymphocytic leukemia (CLL) and its dysregulation in mouse B cells causes a CD5-positive leukemia similar to the aggressive form of human CLLs. To identify oncogenes that cooperate with Tcl1, we performed genetic screen in Eμ−TCL1 mice using Sleeping Beauty transposon-mediated mutagenesis. Analysis of transposon common insertion sites identified 7 genes activated by transposon insertions. Overexpression of these genes in mouse CLL was confirmed by real time reverse transcription-polymerase chain reaction. Interestingly, the main known function of 4 of 7 genes (Nfkb1, Tab2, Map3K14, and Nfkbid) is participation in or activation of the nuclear factor-kB (NF-kB) pathway. In addition, activation of the NF-kB is 1 of main functions of Akt2, also identified in the screen. These findings demonstrate cooperation of Tcl1 and the NF-kB pathway in the pathogenesis of aggressive CLL. Identification cooperating cancer genes will result in the development of combinatorial therapies to treat CLL.

The Role of the BCR Class Expressed by Eμ-TCL1tg Mice and Human CLL

AbstractAbstract 182The B-cell antigen receptor (BCR) plays a critical role in the development and progression of B-cell lymphomas. In chronic lymphocytic leukemia (CLL), the existence of stereotyped heavy-chain complementarity determining regions (HCDR3) suggested that binding of external antigens might play a role in CLL pathogenesis. In contrast, we recently reported that BCRs derived from both human CLL patients and from Eμ-TCL1tg mice have the unique function to induce antigen-independent signaling. This capacity is mediated by the HCDR3 through binding to a BCR internal motif in adjacent BCRs on the same cell (Dühren-von Minden et al., Nature 2012). Mature B cells as well as CLL B cells co-express IgM and IgD with the same HCDR3. In this study, we address the respective roles of these expressed BCR classes in lymphoma pathogenesis in Eμ-TCL1tg mice and in human CLL.By mating Eμ-TCL1tg-mice with IgM−/− mice, which lacks the μ constant heavy domain (μCH) and instead expresses IgD in all developmental stages, we demonstrate a significantly lower frequency of CD19+CD5+IgM+IgDlow lymphoma cells in heterozygous IgM+/−TCL1 mice compared to conventional IgM+/+TCL1 mice (p=0.007). Furthermore, IgM+/−TCL1 mice show a delayed or slowed disease progression compared to TCL1tg mice carrying both IgM alleles. In both TCL1tg mice strains, lymphoma development was exclusively linked to expression of surface IgM, since no IgD single positive lymphoma was detected in IgM+/−TCL1 mice (n=12). TCL1tg mice that lack both μCH alleles showed an accumulation of CD19+CD5+ cells in the spleen at the age of 6 months. According to the genotype of these mice, this population was indeed IgD single positive. However, no further progression could be observed during follow-up to an age of 8 months, indicating a benign form of lymphoproliferation. In contrast to BCRs derived from Eμ-TCL1 mice, analysis of the signaling properties of BCRs derived from IgM−/−TCL1 mice failed to show any autonomous signaling capacity, even when they were expressed as IgM. To address whether IgD in general is able to mediate autonomous signaling reported for TCL1tg- and CLL-derived BCRs, we tested these receptors for autonomous signaling capacity when expressed as IgD. However, expression as IgD led to a complete loss of autonomous signaling capacity in all cases (n=10).In conclusion, whereas autonomous signaling is a characteristic feature of TCL1tg- and CLL-derived BCRs, the pathogenesis of CLL is dependent on the expression of their BCR as the IgM isotype. Expression of IgD-BCRs leads to loss of autonomous signaling capacity, and mice that lack μCH fail to develop malignant lymphoproliferation.To address the question if differential expression of IgD and IgM also had an impact on the clinical behavior of human CLL, we measured the relative expression levels of surface IgD and IgM on circulating lymphoma cells from 67 CLL patients by flow cytometry with simultaneous staining. According to previous reports (Mockridge et al., Blood 2007), unmutated (UM-CLL) cases (n=22) had a higher level of total surface Ig compared to mutated CLL (M-CLL) cases (n=45). Based on our results that IgM is more potent to drive lymphoproliferation, we calculated the ratio of mean fluorescence intensities for IgD over IgM, further called DvM-Score, for every case. A significant difference in the expression pattern as represented by the DvM-Score was observed for UM-CLL and M-CLL (p=0.003) as well as for ZAP70+ and ZAP70- cases (p=0.0002). Both UM-CLL cases as well as ZAP70+ cases show a higher amount of IgM compared to IgD represented by a DvM-Score of <1, whereas the majority of M-CLL and ZAP70− cases express less IgM than IgD and show a DvM-Score of >1. Based on receiver operating characteristics, an DvM cut-off value of 1.15 was identified to optimally discriminate mutational status (AUC: 0.72) and ZAP70 expression (AUC: 0.78). Preliminary data show that CLL samples with a DvM-Score <1.15 had a more aggressive disease course as indicated by a median time to first treatment (TTFT) of 39 months, whereas cases with a DvM-Score of >1.15 show a median TTFT of 154 months (log rank test, p=0.0014).In summary, our results demonstrate an important role of the BCR class, especially with respect to the pathogenetic role of autonomously active IgM BCRs expressed by CLL B cells, for the outcome of the disease. In addition, the DvM score may represent a convenient and novel prognostic marker for CLL. Disclosures:No relevant conflicts of interest to declare.

Potent Anti-Leukemic Activity of a Novel Cdk9 Inhibitor in Preclinical CLL Mouse Models

Abstract 1789Active agents for the treatment of CLL are available. However, CLL eventually progresses to refractory disease. Defects in apoptosis, cytogenetic abnormalities and alterations in non-malignant cells of the micro-environment are likely to be the major causes of therapeutic resistance. Therefore, novel agents are required to abrogate apoptosis blocks and to alter the micro-environment of CLL. One approach targets cyclin-dependent kinases (Cdk). Specifically, Cdk9 promotes initiation and elongation steps in transcription maintaining expression levels of anti-apoptotic proteins with inherently rapid turnover rates. Inhibitors of cyclin-dependent kinases (Cdks) have been previously shown to exert cytotoxic effects on CLL cells by inhibiting Cdk7 and Cdk9. However, inhibitors used in these studies suffer from lack of selectivity, making unambiguous interpretation of results difficult, and leading to unspecific toxicity.Here, we studied the novel Cdk inhibitor LDC-9-A, which exhibits potent and selective inhibitory activity against Cdk9. The in vitro cytotoxicity of LDC-9-A was evaluated after 24 and 48 hours in CLL cases (n=8) and the CLL derived prolymphocytic cell line MEC-1. After a culture period of 48 hours LD50 values ranged between 0.1 and 1.9 μM for primary CLL cells and MEC-1 cells, respectively. Western blot analysis of protein extracts from MEC-1 cells showed that LDC-9-A down-modulates the anti-apoptotic proteins Bcl-2 and Mcl-1 in a dose- and time-dependent manner.In order to study the in vivo anti-CLL activity of LDC-9-A 9–12 month-old transgenic TCL1 mice (n=4) were treated for two consecutive days with an orally bioavailable LDC-9-A formulation (50 mg/kg body weight). Control groups consisted of TCL1 mice receiving vehicle only (n=3) and wild-type control littermates (n=4) receiving LDC-9-A. Numbers of peripheral blood leukemic cells (CD19+CD5+CD3−) and non-malignant T-cells (CD19−CD5+CD3+) were longitudinally evaluated by flow cytometry. Remarkably, LDC-9-A treatment of TCL1 transgenic mice led to a decrease of blood leukemic cells to 6.3±2.0% (mean±SEM) of baseline levels (p<0.0001) two days post treatment cessation. The numbers of T-cells decreased to 15.5±6.6% (p<0.01) in TCL1 mice while the blood T-cells of LDC-9-A treated wild-type controls did only decrease to 49.5±11.6% of baseline levels. Because LDC-9-A was expected to alter cellular Cdk9-mediated anti-apoptotic protein replenishment Bcl-2 levels were determined by flow cytometry analysis. Interestingly, cellular Bcl-2 levels of surviving leukemic cells as well as surviving non-malignant T-cells were significantly higher compared to levels before treatment (1.6- and 2.6-fold, respectively, p<0.05). These data implied that either LDC-9-A was not able to clear cells with high Bcl-2 expression or that LDC-9-A led to a compensatory up-regulation of Bcl-2 in residual cells.Finally, the anti-CLL activity of LDC-9-A was further validated by employing a recently developed adoptive transfer model of primary human CLL cells. In this model NOD/SCID/γcnull (NSG) mice were transplanted with CLL cells and treated with different doses of LDC-9-A for 3–5 sequential days. In line with the data generated with the TCL1 murine CLL model the LDC-9-A treatment of xenografted animals with 60 and 90 mg/kg body weight resulted in an 80–90% reduction of human CLL cell recovery from murine spleens as compared to vehicle treated controls.In summary, we demonstrated effective anti-CLL activity of the novel Cdk9 inhibitor LDC-9-A in two independent preclinical mouse models. The activity of LDC-9-A against CLL cells was achieved at least in part by modulating cellular anti-apoptotic protein levels. Furthermore, LDC-9-A interfered with non-malignant T-cells, which represent an important component of the CLL micro-environment. Disclosures:Eickhoff:Lead Discovery Center GmbH (LDC): Employment. Baumann:Lead Discovery Center GmbH (LDC): Employment. Unger:Lead Discovery Center GmbH (LDC): Employment. Schultz-Fademrecht:Lead Discovery Center GmbH (LDC): Employment. Dürig:Roche: Speakers Bureau; GSK: Speakers Bureau; Santaris Pharma: Consultancy, Research Funding; Celgene: Research Funding.