Mouse Model Of Chronic Lymphocytic Leukemia Research Articles

PD-L1 checkpoint blockade prevents immune dysfunction and leukemia development in a mouse model of chronic lymphocytic leukemia

AbstractBlockade of the programmed cell death 1 (PD-1)/programmed death-ligand 1 (PD-L1) immune checkpoint augments antitumor immunity and induces durable responses in patients with solid cancers, but data on clinical efficacy in leukemias are sparse. Chronic lymphocytic leukemia (CLL) is associated with a tumor-supportive microenvironment and a dysfunctional immune system, as shown by “exhausted” T cells, defective immunologic synapse formation, and immunosuppressive myeloid cells. These defects involve aberrant expression of PD-L1 and are closely mirrored in the Eµ-TCL1 mouse model for CLL. In this study, we treated mice after adoptive transfer of Eµ-TCL1 CLL with PD-L1–blocking antibodies, which prevented CLL development and was accompanied by a reactivation of immune effector functions. This included restoration of mature macrophages and major histocompatibility complex class II–expressing dendritic cells and prevention of aberrant and exhaustion-like T-cell phenotypes. In addition, PD-L1 blockade restored CD8 T-cell cytotoxicity and immune synapse formation and normalized T-cell cytokines and proliferation ex vivo and in vivo. Our data demonstrate that early PD-L1 blockade effectively corrects leukemia-induced immune dysfunction and thus prevents CLL development in mice. Targeting PD-L1/PD-1 interactions should therefore be further explored in clinical studies with CLL patients, ideally in combination with novel compounds to help eliminate CLL.

Mechanisms of PD-L1/PD-1–mediated CD8 T-cell dysfunction in the context of aging-related immune defects in the Eµ-TCL1 CLL mouse model

AbstractT-cell defects, immune suppression, and poor antitumor immune responses are hallmarks of chronic lymphocytic leukemia (CLL), and PD-1/PD-L1 inhibitory signaling has emerged as a major immunosuppressive mechanism. However, the effect of different microenvironments and the confounding influence of aging are poorly understood. The current study uses the Eμ-TCL1 mouse model, which replicates human T-cell defects, as a preclinical platform to longitudinally examine patterns of T-cell dysfunction alongside developing CLL and in different microenvironments, with a focus on PD-1/PD-L1 interactions. The development of CLL was significantly associated with changes in T-cell phenotype across all organs and function. Although partly mirrored in aging wild-type mice, CLL-specific T-cell changes were identified. Murine CLL cells highly expressed PD-L1 and PD-L2 in all organs, with high PD-L1 expression in the spleen. CD3+CD8+ T cells from leukemic and aging healthy mice highly expressed PD-1, identifying aging as a confounder, but adoptive transfer experiments demonstrated CLL-specific PD-1 induction. Direct comparisons of PD-1 expression and function between aging CLL mice and controls identified PD-1+ T cells in CLL as a heterogeneous population with variable effector function. This is highly relevant for therapeutic targeting of CD8+ T cells, showing the potential of reprogramming and selective subset expansion to restore antitumor immunity.

The BAFF receptor TACI controls IL-10 production by regulatory B cells and CLL B cells.

Interleukin (IL)-10-producing B cells (B10 cells) have emerged as important regulatory players with immunosuppressive roles. Chronic lymphocytic leukemia (CLL) B cells also secrete IL-10 and share features of B10 cells, suggesting a possible contribution of CLL B cells to immunosuppression in CLL patients. Factors controlling the emergence of B10 cells are not known. B cell-activating factor of the tumour necrosis factor (TNF) family (BAFF) is critical for B cell maturation and survival, and is implicated in the development and progression of CLL. We sought to investigate the role of BAFF in the emergence of IL-10-producing B regulatory cells in healthy donors and CLL patients. Here, we report that BAFF signaling promotes IL-10 production by CLL B cells in a mouse model of CLL and in CLL patients. Moreover, BAFF-mediated IL-10 production by normal and CLL B cells is mediated via its receptor TACI. Our work uncovered a major targetable pathway important for the generation of regulatory B cells that is detrimental to immunity in CLL.

Selinexor is effective in acquired resistance to ibrutinib and synergizes with ibrutinib in chronic lymphocytic leukemia

AbstractDespite the therapeutic efficacy of ibrutinib in chronic lymphocytic leukemia (CLL), complete responses are infrequent, and acquired resistance to Bruton agammaglobulinemia tyrosine kinase (BTK) inhibition is being observed in an increasing number of patients. Combination regimens that increase frequency of complete remissions, accelerate time to remission, and overcome single agent resistance are of considerable interest. We previously showed that the XPO1 inhibitor selinexor is proapoptotic in CLL cells and disrupts B-cell receptor signaling via BTK depletion. Herein we show the combination of selinexor and ibrutinib elicits a synergistic cytotoxic effect in primary CLL cells and increases overall survival compared with ibrutinib alone in a mouse model of CLL. Selinexor is effective in cells isolated from patients with prolonged lymphocytosis following ibrutinib therapy. Finally, selinexor is effective in ibrutinib-refractory mice and in a cell line harboring the BTK C481S mutation. This is the first report describing the combined activity of ibrutinib and selinexor in CLL, which represents a new treatment paradigm and warrants further evaluation in clinical trials of CLL patients including those with acquired ibrutinib resistance.

Targeting dysfunctional myeloid cells delays disease development and improves immune function in a CLL mouse model (TUM6P.1009)

Abstract Previous in vitro studies have shown that myeloid cells play a key role in apoptosis-resistance in chronic lymphocytic leukemia (CLL). However, their composition and function have not been thoroughly investigated in vivo. Using the Eµ-TCL1 mouse model of CLL, we observed severe skewing of myeloid cell populations along with CLL development. Monocytes and M2-like macrophages infiltrated the peritoneal cavity of leukemic mice. Monocytes expressing high levels of inflammatory factors like IL-10, TNFα and CXCL9 accumulated also in the spleen, the main site of disease, and were severely skewed towards Ly6Clow phenotype. Along with that, the percentage of MHC-IIhi dendritic cells (DCs) and macrophages significantly dropped in the spleen, suggesting monocyte differentiation arrest. Myeloid cell depletion using liposomal clodronate resulted in a significant control of CLL development, repair of innate immune cell phenotypes and partial resolution of systemic inflammation. Also, CLL-induced loss of naive T cells was resolved. CLL-associated monocytes and DCs aberrantly express high PD-L1 levels suggesting their contribution in T cell defects. Importantly, PD-L1 blockade very effectively prevented CLL development and was accompanied with a re-activation of immune effector cells. This included restoration of mature macrophages and MHC-IIhi DCs. Our preclinical data suggests that targeting myeloid cell survival and immunosuppression can serve as a novel strategy for CLL immunotherapy.

A Selective Novel Peroxisome Proliferator-Activated Receptor (PPAR)-α Antagonist Induces Apoptosis and Inhibits Proliferation of CLL Cells In Vitro and In Vivo.

Tumor-specific metabolic changes can reveal new therapeutic targets. Our findings implicate a supporting role for fatty acid metabolism in chronic lymphocytic leukemia (CLL) cell survival. Peroxisome proliferator-activated receptor (PPAR)-α, a major transcriptional regulator of fatty acid oxidation, was recently shown to be upregulated in CLL. To evaluate PPARα as a potential therapeutic target, we developed a highly selective, potent small molecule antagonist of PPARα, NXT629. NXT629 inhibited agonist-induced transcription of PPARα-regulated genes, demonstrating target engagement in CLL cells. Furthermore, NXT629 induced apoptosis of CLL cells even in the presence of a protective microenvironment. To mimic the proliferative lymphoid compartment of CLL, we examined the activity of NXT629 on CLL cells that were stimulated to proliferate in vitro. NXT629 reduced the number of leukemia cells undergoing cell division. In addition, in two xenograft mouse models of CLL (one a model for nondividing and one for dividing CLL), NXT629 reduced the number of viable CLL cells in vivo. Overall, these results suggest that fatty acid metabolism promotes survival and proliferation of primary CLL cells and that inhibiting PPARα gene regulation could be a new therapeutic approach to treating CLL.

PTPROt-mediated regulation of p53/Foxm1 suppresses leukemic phenotype in a CLL mouse model.

The gene encoding PTPROt is methylated and suppressed in Chronic Lymphocytc Leukemia. PTPROt exhibits in vitro tumor suppressor characteristics through the regulation of B-cell receptor signaling. Here, we generated transgenic (Tg) mice with B-cell specific expression of PTPROt. While lymphocyte development is normal in these mice, crossing them with TCL1 Tg mouse model of CLL results in a survival advantage compared to the TCL1 Tg mice. Gene expression profiling of splenic B-lymphocytes before detectable signs of CLL followed by Ingenuity Pathway Analysis revealed that the most prominently regulated functions in TCL1 Tg vs non-transgenic (NTg) and TCL1 Tg vs PTPROt/TCL1 double Tg are the same and also biologically relevant to this study. Further, enhanced expression of the chemokine Ccl3, the oncogenic transcription factor Foxm1 and its targets in TCL1 Tg mice were significantly suppressed in the double Tg mice suggesting a protective function of PTPROt against leukemogenesis. This study also showed that PTPROt mediated regulation of Foxm1 involves activation of p53, a transcriptional repressor of Foxm1, which is facilitated through suppression of B-cell receptor signaling. These results establish the in vivo tumor suppressive function of PTPROt, and identify p53/Foxm1 axis as a key downstream effect of PTPROt-mediated suppression of BCR signaling.

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 Transcription Factor IRF4 Is Crucial for CLL Progression and Regulates Survival and Proliferation in a Microenvironment Related Manner

Introduction: Chronic Lymphocytic Leukemia (CLL) is characterized by an accumulation of CD19+ CD5+ CLL cells in the peripheral blood and the lymphoid compartments. The disease is still incurable despite recent advances in the development of novel therapy concepts (e.g. inhibitors of BCR signaling). Although it is not entirely clear how CLL develops, proliferation centers located in the lymph node and the bone marrow are thought to be the crucial niche that provides the surrounding in which CLL cells originate and re-establish the clone after therapy, leading to relapse in almost 100% of CLL patients. In analogous normal lymphoproliferative environments the transcription factor IRF4 plays an important role in controlling B cell maturation, differentiation, proliferation and survival. A SNP in the human IRF4 3’UTR was linked to CLL susceptibility (DiBernardo Nat.Gen. 2008) and in the mouse model germline deletion of IRF4 in all lineages contributed to CLL development in the New Zealand Black and in the VH11 mouse model (Shukla Blood 2013, Ma J.Biol.Chem. 2013).Methods: CLL samples were collected from the peripheral blood of CLL patients according to the Declaration of Helsinki and with written informed consent. IRF4 mRNA expression analyses were performed in purified CD19+ CLL cells using TaqMan Real Time PCR. In vitro studies were performed in soloculture without stimulation or in coculture systems mimicking the lymph node microenvironment, as previously described (Asslaber Br.J.Haematol. 2013). IRF4 knockdown was performed using lipofection and IRF4 specific siRNAs or non-targeting control siRNAs. IRF4 protein expression was measured by intracellular flow cytometry. Proliferation was detected by Ki-67 intracellular staining. All intracellular stainings were performed after gating on viable CD19+ CD5+ CLL cells. Cell death was assessed by Annexin V / 7AAD staining or using a fixable viability dye.Results: Overall, IRF4 mRNA expression was decreased in CLL patients (N=101) when compared to healthy donors (N=8, P=0.04). In the CLL cohort low IRF4 mRNA expression (cut-off determined by ROC analysis and Youden Index calculation) was significantly correlated with earlier time to treatment (median treatment-free survival: 40 month in the IRF4LOW and 69 month in the IRF4HIGH CLL patient group (P<0.01)). This was proved to be independent of mutation state and other CLL risk factors in multivariate analysis. Using in vitro knockdown of IRF4 in solo- and coculture models we found that lowered expression of IRF4 was associated with increased CLL cell survival and abrogated spontaneous apoptosis (N=11, P<0.01). Moreover, we detected a failure of CLL cells to respond to CD40L signals derived from the microenvironment after IRF4 knockdown, which was associated with abrogated proliferation (N=9, P<0.01). Indeed microenvironmental stimuli (e.g. CD40L ligation or addition of activated T cells) were required to maintain IRF4 expression in vitro (N=17, P < 0.001) which also corresponds to our observation that IRF4 positive CLL cells were highly overrepresented in the CXCR4dim CD5brightCLL population (N = 55, P < 0.0001) which is considered to be the proliferative CLL cell fraction emigrated from the lymph node (Calissano Mol. Med. 2011).Conclusion: We demonstrate that low IRF4 expression is an independent negative prognostic marker for CLL progression. In the microenvironmental context IRF4 plays a dual role, with high expression limiting CLL survival on the one hand but being a crucial mediator of CD40L mediated proliferation on the other hand. While our data suggest that IRF4 is upregulated in response to microenvironmental stimuli the association of low IRF4 levels with bad prognosis suggests that IRF4 levels may serve to limit CLL growth by enhancing cell death responses. The exact mechanisms by which IRF4 impacts CLL pathophysiology is currently being investigated in vivo using B-cell specific IRF4-deficient CLL mouse models. DisclosuresNo relevant conflicts of interest to declare.

Expression of PRMT5 in B-Cell Chronic Lymphocytic Leukemia and Its Significance in Disease Progression and Richter’s Transformation

Richter’s syndrome (RS) represents the transformation of B-cell chronic lymphocytic leukemia (CLL) to a large cell or immunoblastic lymphoma occurring in up to 15% of patients and is associated with poor prognosis and limited treatment options. While RS was first described in 1928, the molecular, genetic and/or epigenetic events that drive CLL B-cell transformation to lymphoma remain poorly characterized. A more comprehensive understanding of the pathogenesis underlying CLL disease progression to lymphoma is needed to (i) reveal potential biomarker(s) to identify CLL patients at risk of transformation and (ii) facilitate the discovery of novel targeted therapeutic approaches for this incurable disease.Epigenetic regulation mediated by lysine- and arginine modifying enzymes play an essential role in tumorigenesis and enhanced nuclear expression of the type II protein arginine methyltransferase, PRMT5, has been associated with increased cell proliferation and survival. Studies on PRMT5 demonstrate its function as an oncogene and its involvement in epigenetic silencing of tumor suppressor and growth regulatory genes in tumor cells; however, its implication in CLL progression is not known. Immunoblot analysis of PRMT5 expression in CLL cells derived from 50 CLL patients with low- (13q) or high-risk (del11q and/or 17p) cytogenetic features revealed no differences in PRMT5 expression among the different cytogenetic groups. However, evaluation of the cellular localization of PRMT5 in 28 patients revealed significantly elevated nuclear PRMT5 levels in CLL cells derived from the high-risk patients. We next evaluated the expression of PRMT5 in CLL cells from patients that developed RS and demonstrate that PRMT5 is overexpressed months to years prior to transformation. This suggests a possible role of PRMT5 overexpression as both a driver event in CLL transformation to DLBCL as well as a potential therapeutic target for intervention. Inhibition of PRMT5 using two small molecule, selective inhibitors (HLCL-61 and HLCL-65) was tested on CLL cells and a significant correlation was observed between HLCL-65 induced cytotoxicity and PRMT5 expression in primary CLL tumor cells. However when patients were separated in low- and high-risk disease groups a significant correlation was observed only in the high-risk group. The effect of inhibition of PRMT5 was also tested in presence or absence of CpG oligonucleotides, a well characterized inducer of CLL cell proliferation. PRMT5 inhibition resulted in significant cytotoxicity and attenuated the survival effect induced by CpG stimulation in CLL cells derived from patients with either low- or high-risk disease. Notably, PRMT5 inhibition markedly interfered with critical pathways important for CLL progression and survival including PI3K, MAPK/ERK as well as cMyc and cJun in CpG-stimulated cells. Additionally, nuclear PRMT5 levels were found to be markedly higher in CpG-stimulated B-cells suggesting a possible role of PRMT5 in the CpG-induced survival pathways.The expression pattern of PRMT5 was also studied in the Eμ-TCL1 transgenic mouse model of CLL. Immunoblot analysis of splenocytes derived from mice at different disease stage revealed a higher level of nuclear PRMT5 in mice with advanced disease. Notably, PRMT5 inhibition in B-cells derived from mice with advanced leukemia induced significant cytotoxicity and reexpression of ST7 a known PRMT5 target gene as well as derepression of miRs implicated in malignant B-cells such as mir-16, miR-106b and miR-223.Together, these findings indicate that PRMT5 contributes to CLL disease progression, and may prove as a candidate prognostic marker identifying CLL patients at risk for transformation; wherein therapeutic strategies aimed at inhibiting PRMT5-driven oncogenic pathways are highly warranted. DisclosuresNo relevant conflicts of interest to declare.

Abstract 4791: The role of microRNA-15a/16 in early B1 cell development in a mouse model of chronic lymphocytic leukemia

Abstract New Zealand Black (NZB) mouse is a de-novo model of chronic lymphocytic leukemia (CLL) that has been studied as a model of B-cell lymphoproliferative disorder. In most human CLL and NZB there is decreased expression of the microRNA miR15a/16. In NZB (and some CLL patients) this is due a point mutation six bases downstream from pre-miR-16 region. In both NZB mouse model and human CLL, the disease is characterized by the presence of a malignant clone of B-1 cells expressing dull levels of CD5 (T cell marker), CD11b (myeloid marker) and B220 (B cell marker). In this report we investigated the molecular pathways that result in increased B-1 lineage development in the mouse model of CLL. Initially we observed that in NZB, the B-1 lineage cells had reduced expression of Pu.1 when compared to NZB B-2 cells. PU.1 is a transcription factor required for B lineage differentiation and PU.1 knockout mice were previously found by others to have only B-1 lineage cell differentiation. To study early B1/B2 divergent cell development pathways in the context of CLL and shed light on the potential malignant cell origin of this disease, we generated induced pluripotent stem cells (iPS) from NZB spleen stromal fibroblasts. Our in vivo and in vitro studies on NZB iPS and hematopoietic progenitors (LSK) differentiation towards B-cell lineage cells followed by multicolor flow based cell sorting revealed a substantial block in the maturation of NZB iPS cells compared to wild type counterparts. Preliminary data using congenic NZB mice with corrected mir-15a/16-1 locus demonstrated that normal levels of miR-15a/16 regulate B-cell differentiation towards a higher expression of PU.1 and B220 (CD45R) and lower levels of CD11b suggestive of an enhanced B2 cell maturation pathway. Our results support the hypothesis that decreased miR-15a plays a role in B1 lymphocyte development bias in the course of B cell maturation by regulating the expression of a number of genes such as B220, PU.1 and CD11b. This work will help further uncover early molecular events which determine the increased B1 versus B2 lineage commitment associated with low levels of miR-15a/16 as an initial lesion in the development of CLL. Citation Format: Chingiz Underbayev, Siddha Kasar, Heba Degheidy, Gerald Marti, Marilyn Lightfoote, Elizabeth Raveche. The role of microRNA-15a/16 in early B1 cell development in a mouse model of chronic lymphocytic leukemia. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4791. doi:10.1158/1538-7445.AM2014-4791

Abstract 1881: New therapeutic options for CLL treatment: Src/c-abl-directed molecular re-engineering of chlorambucil and bendamustine

Abstract B-cell chronic lymphocytic leukemia (CLL) is characterized by actively dividing B-lymphocytes in the lymph nodes and bone marrow, associated with the accumulation of quiescent lymphocytes in the peripheral blood of affected patients. Current treatments for this disease include chemotherapeutic (chlorambucil (CLB), cyclophosphamide, fludarabine) and immunotherapeutic agents (Rituximab, Alemtuzumab) or the combination of immunotherapy with chemotherapeutics drugs. In 2008, the FDA approved bendamustine, a bifunctional agent with a nitrogen mustard moiety and a purine analog, for the treatment of patients with CLL. Signals from the bone marrow and lymph node microenvironments protect CLL lymphocytes from drug-induced apoptosis. During treatment the enzyme-mediated repair of DNA damage can induce resistance to chemotherapeutic drugs. We have previously shown that small molecule inhibitors of c-abl (a key protein of the homologous recombinational repair pathway) such as imatinib, nilotinib or dasatinib, sensitize primary CLL lymphocytes to CLB. We also demonstrated that ZRF4, a combi-molecule designed to target c-abl (through an imatinib moiety) and to induce DNA damage (through a nitrogen mustard moiety), has a more potent cytotoxic effect than the combination of CLB plus imatinib in CLL lymphocytes. Here we report the anticancer effect of three different combi-molecules composed of a chlorambucil moiety and a dasatinib (a dual Src/c-abl inhibitor) moiety compare to the individual components in primary CLL lymphocytes in-vitro. Using MTT assays on CLL lymphocytes from 40 CLL patients, we found that the IC50 (concentration which kills 50% of the cells) of AL748, AL758 and AL816 are significantly lower than the IC50 of CLB when used alone and the combination of CLB with 0.1 μM dasatinib (median value = 1.7 μM, 0.6 μM, 0.9 μM, 11 μM and 3.1 μM respectively). Moreover, our biochemical results suggest that the mechanism of action of AL758, AL816, CLB and CLB plus dasatinib share common downstream targets including inhibition of Src kinase, chemotaxis, and cross-talk with the microenvironment along with induction of DNA damage (p53, p21, comet assay) and apoptosis (AnnexinV, cleaved caspase-3). We also determined, by the MTT assay, the cytotoxic effect of the combi-molecule AL887 composed of a bendamustine moiety and a dasatinib moiety, compare to the individual components in vitro in primary B-lymphocytes from 20 CLL patients. Our results demonstrated that this combi-molecule has a better anticancer activity than bendamustine in combination with 0.1 μM dasatinib (p&amp;lt;0.0005). Dose-limiting toxicity and drug pharmacokinetics are important and limiting factors to take into account for the development of chemotherapy and will be tested in a CLL mouse model. Our results suggest that the combi-molecules AL758, AL816, and AL887 may be useful alternatives for treatment of CLL patients. Citation Format: Lilian Amrein, Anne-Laure Larocque, David Davidson, Lisa Peyrard, Daniel Borrelli, Bertrand Jean-Claude, Lawrence Panasci. New therapeutic options for CLL treatment: Src/c-abl-directed molecular re-engineering of chlorambucil and bendamustine. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1881. doi:10.1158/1538-7445.AM2014-1881

S-15 : Cytokine-driven loss of plasmacytoid dendritic cell function in chronic lymphocytic leukemia

Chronic lymphocytic leukemia (CLL) is characterized by the accumulation of CD5 + CD19 + B cells in the peripheral blood, and in primary and secondary lymphoid organs. A major complication associated with CLL is severe recurrent infections, which are often fatal. Vulnerability to infection is due to a wide variety of immunological defects, yet the initiating events of immunodeficiency in CLL are unclear. Using CLL patient samples and a mouse model of CLL, we have discovered that plasmacytoid dendritic cells (pDCs), which function underpins the activity of effector immune cells critical for anti-viral immunity and anti-tumor responses, are reduced in number and functionally impaired in progressive CLL. As a result, the levels of interferon alpha (IFNa) production, a cytokine critical for immunity, are markedly reduced. Lower pDC numbers with impaired IFNa production was due to the decreased expression of FMS-like tyrosine kinase 3 receptor (Flt3) and Toll-like receptor 9 (TLR9), respectively. Reduced Flt3 expression was reversed using inhibitors of TGF-b and TNF, an effect correlating with a reduction in tumor load. Defects in pDC numbers and function offer new insight into mechanisms underpinning the profound immunodeficiency affecting CLL patients and provide a potentially novel avenue for restoring immunocompetency in CLL.

Mouse models in the study of chronic lymphocytic leukemia pathogenesis and therapy

AbstractMouse models that recapitulate human malignancy are valuable tools for the elucidation of the underlying pathogenetic mechanisms and for preclinical studies. Several genetically engineered mouse models have been generated, either mimicking genetic aberrations or deregulated gene expression in chronic lymphocytic leukemia (CLL). The usefulness of such models in the study of the human disease may potentially be hampered by species-specific biological differences in the target cell of the oncogenic transformation. Specifically, do the genetic lesions or the deregulated expression of leukemia-associated genes faithfully recapitulate the spectrum of lymphoproliferations in humans? Do the CLL-like lymphoproliferations in the mouse have the phenotypic, histological, genetic, and clinical features of the human disease? Here we compare the various CLL mouse models with regard to disease phenotype, penetrance, and severity. We discuss similarities and differences of the murine lymphoproliferations compared with human CLL. We propose that the Eμ-TCL1 transgenic and 13q14-deletion models that have been comprehensively studied at the levels of leukemia phenotype, antigen-receptor repertoire, and disease course show close resemblance to the human disease. We conclude that modeling CLL-associated genetic dysregulations in mice can provide important insights into the molecular mechanisms of disease pathogenesis and generate valuable tools for the development of novel therapies.

Tumor antigen ROR1 targeted drug delivery mediated selective leukemic but not normal B-cell cytotoxicity in chronic lymphocytic leukemia.

Selective cytotoxicity to cancer cells without compromising their normal counterparts pose a huge challenge for traditional drug design. Here we developed a tumor antigen targeted delivery of immunonanoparticle carrying a novel non-immunosuppressive FTY720 derivative OSU-2S with potent cytotoxicity against leukemic B cells. OSU-2S induces activation of protein phosphatase 2A, phosphorylation and nuclear translocation of SHP1S591 and deregulation of multiple cellular processes in chronic lymphocytic leukemia (CLL) resulting in potent cytotoxicity. To preclude OSU-2S mediated effects on these ubiquitous phosphatases in unintended cells and avoid potential adverse effects we developed a OSU-2S targeted delivery immunonanoparticles (2A2-OSU-2S-ILP), that mediated selective cytotoxicity of CLL but not normal B cells through targeting receptor tyrosine kinase ROR1 expressed in leukemic but not normal B cells. Developing a novel spontaneous CLL mouse model expressing human ROR1 (hROR1) in all leukemic B cells, we demonstrate the therapeutic benefit of enhanced survival with 2A2-OSU-2S-ILP in-vivo. The newly developed non-immunosuppressive OSU-2S, its delivery using human CLL directed immunonanoparticles and the novel transgenic mouse model of CLL that expresses hROR1 exclusively in leukemic B cell surface are highly innovative and can be applied to CLL and other ROR1+ malignancies including mantle cell lymphoma and acute lymphoblastic leukemia.

Cytokine-driven loss of plasmacytoid dendritic cell function in chronic lymphocytic leukemia.

Chronic lymphocytic leukemia (CLL) is characterized by the accumulation of CD5+CD19+ B cells in the peripheral blood, and in primary and secondary lymphoid organs. A major complication associated with CLL is severe recurrent infections, which are often fatal. Vulnerability to infection is due to a wide variety of immunological defects, yet the initiating events of immunodeficiency in CLL are unclear. Using CLL patient samples and a mouse model of CLL, we have discovered that plasmacytoid dendritic cells (pDCs), which underpin the activity of effector immune cells critical for anti-viral immunity and anti-tumor responses, are reduced in number and functionally impaired in progressive CLL. As a result, the levels of interferon alpha (IFNα) production, a cytokine critical for immunity, are markedly reduced. Lower pDC numbers with impaired IFNα production was due to the decreased expression of FMS-like tyrosine kinase 3 receptor (Flt3) and Toll-like receptor 9 (TLR9), respectively. Reduced Flt3 expression was reversed using inhibitors of TGF-β and TNF, an effect correlating with a reduction in tumor load. Defects in pDC numbers and function offer a new insight into mechanisms underpinning the profound immunodeficiency affecting CLL patients and provide a potentially novel avenue for restoring immuno-competency in CLL.

Bruton's tyrosine kinase (BTK) function is important to the development and expansion of chronic lymphocytic leukemia (CLL)

AbstractChronic lymphocytic leukemia (CLL) is characterized by constitutive activation of the B-cell receptor (BCR) signaling pathway, but variable responsiveness of the BCR to antigen ligation. Bruton's tyrosine kinase (BTK) shows constitutive activity in CLL and is the target of irreversible inhibition by ibrutinib, an orally bioavailable kinase inhibitor that has shown outstanding activity in CLL. Early clinical results in CLL with other reversible and irreversible BTK inhibitors have been less promising, however, raising the question of whether BTK kinase activity is an important target of ibrutinib and also in CLL. To determine the role of BTK in CLL, we used patient samples and the Eμ-TCL1 (TCL1) transgenic mouse model of CLL, which results in spontaneous leukemia development. Inhibition of BTK in primary human CLL cells by small interfering RNA promotes apoptosis. Inhibition of BTK kinase activity through either targeted genetic inactivation or ibrutinib in the TCL1 mouse significantly delays the development of CLL, demonstrating that BTK is a critical kinase for CLL development and expansion and thus an important target of ibrutinib. Collectively, our data confirm the importance of kinase-functional BTK in CLL.

Targeting chronic lymphocytic leukemia using CIGB-300, a clinical-stage CK2-specific cell-permeable peptide inhibitor

Chronic lymphocytic leukemia (CLL) remains an incurable malignancy, urging for the identification of new molecular targets for therapeutic intervention. CLL cells rely on overexpression and hyperactivation of the ubiquitous serine/threonine protein kinase CK2 for their viability in vitro. CIGB-300 is a cell-permeable selective CK2 inhibitor peptide undergoing clinical trials for several cancers. Here, we show that CIGB-300 promotes activation of the tumor suppressor PTEN and abrogates PI3K-mediated downstream signaling in CLL cells. In accordance, CIGB-300 decreases the viability and proliferation of CLL cell lines, promotes apoptosis of primary leukemia cells and displays antitumor efficacy in a xenograft mouse model of human CLL. Our studies provide pre-clinical support for the testing and possible inclusion of CK2 inhibitors in the clinical arsenal against CLL.

Effective Inhibition Of Tumor Microenvironment Interactions In CLL Patients Treated With The BTK Inhibitor Ibrutinib Results In Sustained Inhibition Of Tumor Proliferation and Survival Pathways

▪Chronic lymphocytic leukemia (CLL) cells depend on microenvironmental factors for proliferation and survival. In particular, tissue resident CLL cells show an increase in both B-cell receptor (BCR) and NF-κB signaling; pathways known to regulate survival, proliferation and migration of CLL cells. One key signaling molecule in this pathway is Bruton's tyrosine kinase (BTK) that is activated directly downstream of the BCR and known to be up-regulated in CLL cells (Herman et al., Blood 2011). Ibrutinib, a covalent BTK inhibitor currently in clinical trials for CLL, has been shown to induce apoptosis and inhibit proliferation and tumor burden both in vitro and in mouse models of CLL (Herman et al., Blood 2011; Ponader et al., Blood 2012, Herman et al., Leukemia 2013). Recently, in a multicenter study, ibrutinib has been shown to induce objective clinical responses and reduce lymphadenopathy in the majority of patients, regardless of the presence of adverse prognostic markers (Byrd et al., NEJM 2013).It has been shown that CLL cells in the lymph node and bone marrow microenvironments demonstrate higher levels of BCR and NF-κB signaling as well as increased cell activation and proliferation (Herishanu et al., Blood 2011). We therefore sought to determine the in vivo effect of ibrutinib on tumor cell activation and proliferation in these microenvironmental niches. We have previously demonstrated that ibrutinib inhibits BCR and NF-κB in the lymph node microenvironment within the first 24 hours after initiation of therapy (Herman et al., ASH 2012). We here expand our previous results by evaluating the long term effects of ibrutinib in the tissue compartment. Because repeat sampling of lymph node tissue on therapy is not practical, we assessed changes in the bone marrow compartment. We obtained bone marrow aspirates pre-treatment and after two cycles of ibrutinib (Day 56). We first evaluated the BCR gene signature using a previously validated set of BCR regulated genes (Herishanu et al., 2011). We found that BCR signaling was significantly inhibited in CLL cells sampled from the bone marrow in 8/8 patients evaluated (P = 0.01). Similarly we also found inhibition of the NF-κB gene signature in all patients evaluated (P = 0.01). In fact, every patient evaluated demonstrated a reduction in both signatures, but there was substantial variation among patients in the extent to which these pathways were inhibited. This variability did not appear to correlate with clinical and prognostic factors, such as IGHV mutation status, deletion of chromosome 17p, or prior treatment status. However, the degree of inhibition of NF-κB signaling was strongly correlated with the degree of inhibition of BCR signaling (r = 0.96, P < 0.001), suggesting that the BCR (and/or a second equally BTK dependent pathway) plays a major role in activating NF-κB also in bone marrow resident CLL cells. To confirm our results we next evaluated the phosphorylation of proteins activated downstream of the BCR. We found that CLL cells showed a significant reduction in both PLCγ2 and ERK phosphorylation (mean reduction 52.9% and 71.2%, respectively; P < 0.01). Next, using the proliferation marker Ki67 we found a significant reduction in tumor proliferation in the bone marrow on ibrutinib (from a median of 6.6% KI67+ CLL cells pre-treatment to 1.1% on Day 56, P = 0.003). Lastly, we also found a significant reduction in the cell surface expression of the activation markers CD69 and CD86 (mean reduction of 57% (P = 0.001) and 82% (P = 0.03), respectively).In conclusion, our data demonstrate that ibrutinib effectively inhibits BCR, NF-κB, and ERK signaling. This occurs very quickly as demonstrated in the lymph node and is sustained on treatment as shown in the bone marrow. The strong and sustained reduction in proliferation and activation of CLL cells in the tissue microenvironment suggests that BTK is indeed a central hub mediating the nourishing and protective effects of the tumor microenvironment.This work was supported by the Intramural Research Program of NHLBI, NIH. We thank our patients for donating blood and tissue samples to make this research possible. We acknowledge Pharmacyclics for providing study drug. Disclosures:Off Label Use: Ibrutinib in chronic lymphocytic leukemia.

Regulatory B Cells (B10 Cells) in Human Disease

B cells have been historically characterized as positive regulators of immune responses, but can also influence immune responses through numerous mechanisms other than antibody production. In one example, we and others have functionally identified a rare but specific subset of IL-10-competent regulatory B cells in humans and mice by their ability to secrete IL-10. We have labeled these B cells as “B10 cells” to identify them as the exclusive source of B-cell IL-10. The capacity of B10 cells to produce IL-10 is central to their ability to negatively regulate inflammation and autoimmune disease, as well as adaptive and innate immune responses. Rare B10 cell numbers often increase during inflammation and autoimmunity, particularly in some patients with active disease and autoimmune-prone mice. Nonetheless, the in vivo expansion of B10 cells in mice and patients responding to inflammation is inadequate to effectively control disease. However, the adoptive transfer of 1-2x105 spleen B10 cells from naïve or antigen-primed mice significantly inhibits disease initiation in mouse models of inflammation, autoimmune disease, and contact hypersensitivity. B10 cells are not overtly immunosuppressive, but instead have antigen-specific regulatory functions. Appropriate B-cell antigen receptor specificity and signals are required to drive B10 cell development and acquisition of IL-10 competence under physiological conditions in vivo. Following antigen-specific B10 cell development, their maturation into functional IL-10-secreting effector cells in vivo requires IL-21 and CD40-dependent cognate interactions with antigen-specific T cells. These critical checkpoints are likely to direct localized B10 cell IL-10 production to blunt antigen-specific T-cell responses during cognate B10:T cell interactions. These findings likely explain how antigen-specific B10 cell-effector function can exert such potent in vivo effects and selectively inhibit antigen-specific T-cell function during inflammation and autoimmunity without untoward immunosuppression. Human B10 cells are likely to have similar in vivo regulatory activities. In support of this, collaborative studies with Brice Weinberg and others have shown that human blood B10 cells and malignant cells from 90 percent of patients with chronic lymphocytic leukemia (CLL) share similar cell surface phenotypes and the capacity to express IL-10. Mouse B10 cells and malignant cells in the Tcl-1 transgenic mouse model of CLL also share similar phenotypes and the capacity to express IL-10. Of importance, CLL cell production of IL-10 appears to be dynamically regulated in mice and CLL patients are frequently immunosuppressed with abnormalities in both humoral and cellular immunity. Thus, B10 cells and CLL cells share regulatory properties. Insights into the molecular pathways that regulate antigen-specific B10 cell function in vivo have led to the development of an in vitro culture system that expands functionally-active mouse B10 cells (B10 effector cells) ex vivo by 4 million-fold. B10 effector cells resulting from this culture system secrete IL-10, retain their antigen-specific regulatory functions, and have demonstrated potent anti-inflammatory effects that effectively treat mice with established autoimmune disease. In mice, this culture system allows the generation of sufficient B10 effector cells from one mouse to treat 21,000 syngeneic mice with established disease. Thus, autologous B10 effector cells may eventually provide a new therapy that successfully treats patients with severe and refractory autoimmune disease, particularly those individuals for whom effective drugs have not been identified, as well as those who experience transplant rejection. Disclosures:Tedder:Angelica Therapeutics, Inc.: Consultancy, Equity Ownership; National Institutes of Health grants AI56363 and AI057157: Research Funding; Lymphoma Research Foundation: Research Funding.