Cell Chronic Lymphocytic Leukemia Cells Research Articles

Loss of AID exacerbates the malignant progression of CLL

Activation-induced cytidine deaminase (AID) has been implicated as both a positive and a negative factor in the progression of B cell chronic lymphocytic leukemia (CLL), but the role that it plays in the development and progression of this disease is still unclear. We generated an AID knockout CLL mouse model, AID−/−/Eμ-TCL1, and found that these mice die significantly earlier than their AID-proficient counterparts. AID-deficient CLL cells exhibit a higher ER stress response compared to Eμ-TCL1 controls, particularly through activation of the IRE1/XBP1s pathway. The increased production of secretory IgM in AID-deficient CLL cells contributes to their elevated expression levels of XBP1s, while secretory IgM-deficient CLL cells express less XBP1s. This increase in XBP1s in turn leads AID-deficient CLL cells to exhibit higher levels of B cell receptor signaling, supporting leukemic growth and survival. Further, AID−/−/Eμ-TCL1 CLL cells downregulate the tumor suppressive SMAD1/S1PR2 pathway and have altered homing to non-lymphoid organs. Notably, CLL cells from patients with IgHV-unmutated disease express higher levels of XBP1s mRNA compared to those from patients with IgHV-mutated CLL. Our studies thus reveal novel mechanisms by which the loss of AID leads to worsened CLL and may explain why unmutated CLL is more aggressive than mutated CLL.

Abstract 142: Loss of AID exacerbates the malignant progression of CLL

Abstract Activation-induced cytidine deaminase (AID) is an enzyme that has been implicated as both a positive and a negative factor in the progression of B cell chronic lymphocytic leukemia (CLL), but the role that it plays in the development and progression of this disease is still unclear. Higher AID expression in patients has been correlated with a worse prognosis, but patients with mutated immunoglobulin (IgHV) genes, which requires AID activity, have a better prognosis. To elucidate the role of AID in leukemic progression, we generated an AID-deficient mouse model of CLL, AID-/-/Eμ-TCL1, and found that these mice die significantly earlier than their AID-proficient counterparts. AID-/-/Eμ-TCL1 mice have higher percentages of germinal center B cells and increased infiltration of CLL cells into the lungs, liver, and peritoneal cavity. Both AID-deficient B cells and CLL cells downregulated the tumor suppressive Smad1/S1PR2 signaling pathway, which normally restrains expansion of the germinal center, potentially leading to altered proliferation and homing of leukemia cells. Furthermore, AID-/-/Eμ-TCL1 CLL cells exhibited higher levels of B cell receptor (BCR) signaling, supporting their growth and survival. Upregulated BCR signaling led us to investigate the IRE1α/XBP1s pathway in these cells, which is necessary to support proper BCR signaling and is upregulated in CLL cells. IRE1α is an endoplasmic reticulum (ER) stress sensor that cleaves XBP1 mRNA to produce the functional transcription factor XBP1s. XBP1s promotes synthesis of lipids and chaperones necessary for BCR signaling, and inhibition of this pathway compromises BCR signaling and CLL survival. Compared to Eμ-TCL1 controls, AID-/-/Eμ-TCL1 CLL cells indeed respond to various ER stressors with a higher ER stress response, particularly through the IRE1α/XBP1s pathway, resulting in cells that are more resistant to the stressed conditions within the tumor microenvironment. Mechanistically, this upregulation in the ER stress response cannot be solely attributed to an accelerated leukemic progression, as B cells from AID-/- mice also express increased levels of XBP1s and are more responsive to treatment with pharmacological ER stressors. These studies are translatable to human CLL, as shRNA knockdown of AID in the CLL cell line WaC3 resulted in an increased ER stress response, and CLL cells from patients with IgHV-unmutated disease express higher levels of XBP1 mRNA than those from patients with mutated CLL. Additionally, treatment of both mouse and human CLL cells with a combination of the S1PR2 agonist CYM-5520 and the IRE1 inhibitor B-I09 led to improved induction of apoptosis, revealing a novel way to treat CLL. Overall, these results demonstrate multiple pathways through which the loss of AID leads to worsened CLL and may explain why IgHV-unmutated CLL has such a high proliferative rate that requires therapeutic intervention. Citation Format: Avery C. Lee, Sai R. Pingali, Javier A. Pinilla-Ibarz, Chih-Hang A. Tang, Chih-Chi A. Hu. Loss of AID exacerbates the malignant progression of CLL [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 142.

Interleukin-10 suppression enhances CD8+ T cell responses to checkpoint blockade immunotherapy in Chronic Lymphocytic Leukemia

Abstract T-cell-based immunotherapy revolutionized the treatment of immune suppressive cancers. Blocking T cell inhibitory receptors such as programmed death 1 (PD1) or their ligands (like PD-L1) enhances antitumor T cell responses. However, many cancers employ multiple methods of immune suppression, including the production of potent regulatory cytokines like Interleukin-10 (IL-10). B cell Chronic Lymphocytic Leukemia (CLL) is one such B cell malignancy that elicits widespread immune dysfunction including numerous functional deficits in T cells. Human and mouse CLL cells (Eμ-Tcl1) produce IL-10 and PD-L1, which dampen antitumor immunity. Since immune checkpoint blockade experienced limited success in CLL, we investigated IL-10 suppression as a novel combination therapy. Exogenous and CLL-derived IL-10 decreased human and mouse T cell functionality in vitro. Further, CLL-derived IL-10 can be blocked with antibodies or can be reduced with small molecule inhibitors to improve CD8+ T cell functionality in vivo. CLL IL-10 production depends on the transcription factor Sp1. Hence, we utilized a novel analogue of the Sp1 inhibitor mithramycin to reduce CLL IL-10 secretion. IL-10 inhibition improved responses to anti-PD-L1 therapy, with increased survival and decreased CLL burden compared to anti-PD-L1 alone. This method improved CD8+ T cell proliferation, effector-memory cell frequency, and interferon-γ production. CD8+ T cells were also more abundant, with fewer exhausted T cells even at very advanced stages of disease. Current cancer therapies do not target IL-10 and our studies provide evidence for targeting IL-10 to increase the efficacy of T cell immunotherapies in human CLL, and possibly other cancers with T cell suppression.

Enhanced Expression of miR-181b in B Cells of CLL Improves the Anti-Tumor Cytotoxic T Cell Response.

Simple SummaryLow expression of miR-181b in chronic lymphocytic leukemia (CLL) is linked to progression and cell death resistance. Patients with the progression of the disease show immune dysfunction. We aim at studying whether miR-181b is also involved in this process. We demonstrate that miR-181b can be increased in CLL cells by crosstalk with CD40L+ T cells, enhancing the maturation of CD8+ T cells in cytotoxic T lymphocytes (CTL) and, in turn, the anti-tumor cytotoxic T cell response in in vitro and in vivo models. These results demonstrate a role of the miR-181b in the immune response against tumor and envisage a therapeutic action of miR-181b in a specific milieu surrounding CLL cells.The clinical progression of B cell chronic lymphocytic leukemia (CLL) is associated with immune cell dysfunction and a strong decrease of miR-181b-5p (miR-181b), promoting the death of CLL cells. Here we investigated whether the reduction of miR-181b impairs the immune response in CLL. We demonstrate that activated CD4+ T cells increase miR-181b expression in CLL through CD40–CD40L signaling, which enhances the maturation and activity of cytotoxic T cells and, consequently, the apoptotic response of CLL cells. The cytotoxic response is facilitated by a depletion of the anti-inflammatory cytokine interleukin 10, targeted by miR-181b. In vivo experiments in NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ mice confirmed that miR-181b promotes the apoptotic death of CLL cells only when functional T cells are restored. Overall, our findings suggest that the reinstatement of miR-181b in CLL cells could be an exploitable adjuvant therapeutic option for the treatment of CLL.

ROR1 expression in chronic lymphocytic leukemia and the effect of CD5

B cell chronic lymphocytic leukemia (CLL) is characterized by accumulation of monoclonal CD5+ mature B cells. The expression of CD5 plays a role in the malignant behavior of CLL cells via controlling the expression of some genes that enhance the expression of; apoptosis inhibitors BCL-2, NF-κB, Wnt, and cytokine. By being naturally phosphorylated on tyrosines, CD5 is chronically activated in CLL cells. Moreover, B CLL cell also characterized by the expression of ROR1. In this study we wanted to know if CD5 has an effect on the surface expression of ROR1 in CLL cells via comparing the mean fluorescence intensity (MFI) between CD5 and ROR1.Therefore, A cross-sectional study of immune-phenotype results for 50 randomly selected patients diagnosed with CLL in the period from June 2014 till Jan.2016 was performed. 28 cases were excluded because ROR1 test was not performed for them. The study was carried out on 22 patients. The correlation between CD5 MFI and ROR1 MFI was assessed using Spearman’s correlation. We found that in 21 out of 22 cases the correlation was significant with P < 0.001 and correlation coefficient (rs) of 0.788. This study gives us a hint that CD5 might have an effect on the expression of ROR1 in CLL lymphocytes. This finding needs to be supported by experimental study looking at the mechanism by which CD5 has such an effect.

Approaches to reverse interleukin-10 mediated immunosuppression in chronic lymphocytic leukemia

Abstract B cell Chronic Lymphocytic Leukemia (CLL), the most common leukemia in the western world, is characterized by an accumulation of abnormal B cells in the blood and lymphoid organs. Broad immunosuppression is a serious complication for these patients, with infections and secondary cancers recognized as the leading causes of morbidity and mortality. Although the immune suppression is in part due to clonotypic CLL B cells crowding immune organs, immune responses may also be suppressed by the production of soluble mediators. We and others found that CLL cells produce interleukin-10 (IL-10), a cytokine that suppresses T cell effector function. We hypothesized that this IL-10 may reduce anti-tumor T cell responses. In our studies we use human cells as well as the Eμ-Tcl1 mouse model for U-CLL, in which the oncogene Tcl1 is expressed under the immunoglobulin VH promoter and μ-enhancer. Previously, we found that IL-10R−/− T cells control CLL growth better than WT T cells in the Eμ-Tcl1 adoptive transfer model. We also saw that CLL IL-10 production is dependent on BCR signaling and activation of the transcription factor Sp-1. IL-10 production can be blocked by mithramycin (MTM), an Sp-1 inhibitor. However, MTM is not used routinely as an anti-cancer agent due to its short half-life and associated toxicities, so we prepared and tested novel analogues of MTM to reduce these effects. One of these analogues, MTM23, shows promise as it has similar potency to MTM, but does not interfere with T cell gamma-interferon production or viability. This approach is novel as current therapies for CLL do not target IL-10, or address the immunosuppression seen in CLL patients. IL-10 blockade could therefore be a strategy for harnessing anti-tumor T cells to treat CLL in humans.

L-selectin controls trafficking of chronic lymphocytic leukemia cells in lymph node high endothelial venules in vivo

AbstractB-cell chronic lymphocytic leukemia (CLL) is the most common leukemia in adults. Lymph nodes (LNs) are sites of malignant proliferation and LN enlargement is associated with poor prognosis in the clinics. The LN microenvironment is believed to favor disease progression by promoting CLL cell growth and drug resistance. A better understanding of the mechanisms regulating trafficking of CLL cells to LNs is thus urgently needed. Here, we studied the first step of CLL cell migration to LNs, their interaction with high endothelial venules (HEVs), specialized blood vessels for lymphocyte extravasation in lymphoid organs. We observed that the density of HEV blood vessels was increased in CLL LNs and that CD20+ CLL cells accumulated within HEV pockets, suggesting intense trafficking. We used intravital imaging to visualize the behavior of human CLL cells within the mouse LN microcirculation, and discovered that CLL cells bind to HEVs in vivo via a multistep adhesion cascade, which involves rolling, sticking, and crawling of the leukemic cells on the endothelium. Functional analyses revealed that the lymphocyte homing receptor L-selectin (CD62L) is the key factor controlling the binding of CLL cells to HEV walls in vivo. Interestingly, L-selectin expression was decreased on CLL cells from patients treated with idelalisib, a phosphoinositide-3-kinase δ inhibitor recently approved for CLL therapy. Interference with L-selectin–mediated trafficking in HEVs could represent a novel strategy to block dissemination of CLL cells to LNs and increase the efficacy of conventional therapy.

Investigation of T Cell Subsets and Germinal Center Reaction in a B Cell Malignancy Model of Mice Deficient in the Lymphoma Hyermutable Gene Rhoh

Rho GTPases are well known regulators of actin dynamics, gene transcription, kinase pathways and cell cycle progression. RhoH, a hematopoietic specific, GTPase-deficient Rho GTPase was first defined as a hypermutable gene in diffuse large B cell lymphoma (Pasqualucci et al., 2001), although its role in the pathobiology of B cell malignancies still remains unclear. Subsequently, RhoH has been implicated in TCR signaling and T cell development in humans and mice. As RhoH is required for activation and localization of ZAP70 and LCK to the immune synapse, knockout of RhoH results in T cell deficiency (Gu et al., 2006). However, despite a profound T cell defect in Rhoh-/- mice, we previously noted an apparent paradoxical delay in disease onset in the Em-TCL1Tg murine model of B cell chronic lymphocytic leukemia (CLL) after deletion of RhoH. We previously demonstrated that this is partly due to Rhoh-/- CLL cell-intrinsic changes resulting in impaired access to supportive niches and defective microenvironmental interactions (Troeger et al., 2012). However, there is accumulating evidence that progressive immune dysregulation also plays an important role in CLL progression. Specifically, an increase in circulating follicular helper T cells (Tfh), inverted CD4:CD8 ratios, a predominance of a memory T cell phenotype, defective T cell motility and an impaired immunological synapse formation have been reported in CLL patients and Em-TCL1Tg mice (Hofbauer et al., 2011). Interestingly, the immunomodulatory drug lenalidomide has proven effective in modulating CLL-associated changes in T cell function and altered Rho GTPase activity (Ramsay et al., 2013) and we have previously demonstrated that lenalidomide treatment of CLL cells in vivo and in vitro resulted in decreased RhoH expression, suggesting that Rho GTPases are involved in T cell- B cell/CLL interactions. Moreover, lenalidomide treatment has been shown to restore immune synapse formation and T cell function in CLL patients indicating that RhoH may similarly modulate T cell- B cell crosstalk including modified LFA1 signaling.Here we aimed to assess the impact of RhoH on the germinal center reaction and changes in T cell populations over time in knock-out mice and a murine model of CLL. We demonstrate that RhoH is required for normal germinal center (GC) formation and induction of T cell dependent B cell responses in vivo. Thus, while IgM levels were only mildly reduced in Rhoh-/- mice, these animals exhibited significantly reduced IgG1 serum levels 21 days after TNP-KLH treatment (WT vs. Rhoh-/-mice: IgG1 3706951ng/ml +/-871537 vs 122176ng/ml +/-14006; mean+/- SEM; p=0.01), indicating a defect in immunoglobulin class switching.In keeping with these observations, we detected a severe defect in CXCR5+ Tfh cells in the spleens (WT vs Rhoh-/- mice: 10.09%+/-1.23 vs. 1.71%+/-0.45; mean+/- SEM; p=0.01) and peripheral blood (WT vs Rhoh-/- mice: 4.69%+/-0.51 vs 0.36% +/-0.13) of young Rhoh-/- mice, and a profound defect in both naïve CD4+ and CD8+ T cells. However, while over time in mice with CLL disease CD8+ activated effector memory T cells expanded in Em-TCL1Tg;Rhoh-/- to levels comparable to WT mice, deficiency in CD4+ Th cells persisted in Rhoh-/- animals. As it has been shown that CLL cell proliferation depends on Th cell support (Plander et al., 2009), we next aimed to assess the impact of the T cell phenotype on disease progression by performing adoptive transfer experiments. After 6 weeks, recipients of WT CLL cells demonstrated a significantly accelerated disease progression compared to those injected with Rhoh-/- CLL cells (WT vs. Rhoh-/- recipients:238.1+/-65.8 K/µl vs 195.2+/-54.8 K/µl WT CLL cells; and 2.3+/-1.4 K/µl vs 5.9+/-2.5 K/µl Rhoh-/- CLL cells) and accordingly also demonstrated improved survival (survival probability: WT CLL: 0.4+/-0.15 vs. 0.3+/-0.14 and Rhoh-/- CLL: 0.75+/-0.13 vs. 0.82+/-0.12; p<0.05).These data clarify that the lack of CD4+ Th cell support and impaired GC reaction due to RhoH-deficiency have little impact on disease progression in this model of CLL. The findings further confirm that the slower disease progression and improved survival observed in the murine model of Rhoh-/- CLL is mainly mediated by cell autonomous characteristics of the CLL cells and a sufficiently sustained immune surveillance by CD8+ T cells. Thus, inhibition of RhoH may represent an attractive tool for future targeted therapies in CLL. DisclosuresNo relevant conflicts of interest to declare.

Inhibition of ER stress-associated IRE-1/XBP-1 pathway reduces leukemic cell survival.

Activation of the ER stress response is associated with malignant progression of B cell chronic lymphocytic leukemia (CLL). We developed a murine CLL model that lacks the ER stress-associated transcription factor XBP-1 in B cells and found that XBP-1 deficiency decelerates malignant progression of CLL-associated disease. XBP-1 deficiency resulted in acquisition of phenotypes that are disadvantageous for leukemic cell survival, including compromised BCR signaling capability and increased surface expression of sphingosine-1-phosphate receptor 1 (S1P1). Because XBP-1 expression requires the RNase activity of the ER transmembrane receptor IRE-1, we developed a potent IRE-1 RNase inhibitor through chemical synthesis and modified the structure to facilitate entry into cells to target the IRE-1/XBP-1 pathway. Treatment of CLL cells with this inhibitor (B-I09) mimicked XBP-1 deficiency, including upregulation of IRE-1 expression and compromised BCR signaling. Moreover, B-I09 treatment did not affect the transport of secretory and integral membrane-bound proteins. Administration of B-I09 to CLL tumor-bearing mice suppressed leukemic progression by inducing apoptosis and did not cause systemic toxicity. Additionally, B-I09 and ibrutinib, an FDA-approved BTK inhibitor, synergized to induce apoptosis in B cell leukemia, lymphoma, and multiple myeloma. These data indicate that targeting XBP-1 has potential as a treatment strategy, not only for multiple myeloma, but also for mature B cell leukemia and lymphoma.

Validating The Prognostic Significance Of FCRL2 In Predicting IGHV Mutation Status, Clinical Disease Progression, and Survival In CLL

B cell chronic lymphocytic leukemia (CLL) can be stratified into indolent and aggressive subtypes according to the degree of somatic hypermutation evident in the immunoglobulin heavy chain variable region gene (IGHV). Despite some discordance with IGHV mutation status, both CD38 and ZAP-70 are routinely analyzed in clinical practice and are considered useful prognostic factors for segregating patients with aggressive-unmutated CLL (U-CLL). Members of the Fc receptor-like (FCRL1-5) family have immunoregulatory function and are preferentially expressed by B cells. Their distinct distribution pattern among subsets in healthy donors suggests that the FCRLs may also have useful diagnostic and/or therapeutic potential in B lineage malignancies. Previous work employing a receptor-specific monoclonal antibody (mAb-7F2) and an indirect staining method demonstrated that FCRL2 is particularly upregulated on the surface of indolent-mutated CLL (M-CLL) cells. Among 107 CLL samples, 52 U-CLL and 55 M-CLL, FCRL2 overlapped with IGHV status with 94.4% concordance, relative to 76.6% for CD38 (cutoff of 30%), and 80.4% for ZAP-70 according to the T/B ratio (cutoff of 4.0) or 74.8% by the % method (cutoff of 20%). Because several years have passed since our initial analysis using this reagent, we updated the clinical information to reassess the relationship among these prognostic parameters with FCRL2 staining and to further determine the usefulness of this biomarker in CLL. Similar to IGHV status [Hazard ratio (HR)=5.03], FCRL2 could also, but more powerfully, predict survival (HR=8.58). Both these features were superior to CD38 (HR=1.29) and ZAP-70 by either the T/B ratio (HR=1.42) or % (HR=1.04) approaches. The median survival for FCRL2 positive cases was 26.2 years and for FCRL2 negative donors was 12.7 years. With regard to disease progression, FCRL2 positive cases had a median treatment free interval of 13.5 years, whereas for FCRL2 negative donors it was only 3.2 years. Given these promising results, we pursued optimizing FCRL2 detection and assayability by flow cytometry to validate its clinical utility for diagnostic and prognostic purposes in CLL. A screen of FCRL2-specific mAbs led to the identification of a novel subclone termed 3E11, which by epitope mapping reacted with a site discrete from that of 7F2. The binding kinetics of these two mAbs also differed. According to surface plasmon resonance studies using an FCRL2-Fc recombinant protein, 3E11 possessed ∼2.5X better nanomolar affinity and a slower dissociation rate than 7F2. The superiority of 3E11 was also evidenced by its relatively stronger surface staining reactivity in flow cytometry analyses. To achieve a one-step staining strategy, 3E11 was directly conjugated with PE. Use of this PE-labeled reagent demonstrated brighter and more selective FCRL2 reactivity on M-CLL versus U-CLL (N=30) cells using either the post-Ficoll or whole blood-lysis preparation methods. Importantly, its reactivity was also stable over time (Concordance Correlation Coefficient of 0.9). As a promising CLL biomarker capable of selectively detecting the MT-CLL subtype and predicting disease progression and survival, the current findings demonstrate the practicability and reliability of a newfound mAb for validating the prognostic value of FCRL2 in CLL. Disclosures:LI:University of Alabama at Birmingham: Filed Patent, Filed Patent Patents & Royalties. Davis:NIH an University of Alabama at Birmingham: Patent Filed, Patent Filed Patents & Royalties, Research Funding.

A mutated B cell chronic lymphocytic leukemia subset that recognizes and responds to fungi

B cell chronic lymphocytic leukemia (CLL), the most common leukemia in adults, is a clonal expansion of CD5(+)CD19(+) B lymphocytes. Two types of CLLs are being distinguished as carrying either unmutated or somatically mutated immunoglobulins (Igs), which are associated with unfavorable and favorable prognoses, respectively. More than 30% of CLLs can be grouped based on their expression of stereotypic B cell receptors (BCRs), strongly suggesting that distinctive antigens are involved in the development of CLL. Unmutated CLLs, carrying Ig heavy chain variable (IGHV) genes in germline configuration, express low-affinity, poly-, and self-reactive BCRs. However, the antigenic specificity of CLLs with mutated IGHV-genes (M-CLL) remained elusive. In this study, we describe a new subset of M-CLL, expressing stereotypic BCRs highly specific for β-(1,6)-glucan, a major antigenic determinant of yeasts and filamentous fungi. β-(1,6)-glucan binding depended on both the stereotypic Ig heavy and light chains, as well as on a distinct amino acid in the IGHV-CDR3. Reversion of IGHV mutations to germline configuration reduced the affinity for β-(1,6)-glucan, indicating that these BCRs are indeed affinity-selected for their cognate antigen. Moreover, CLL cells expressing these stereotypic receptors proliferate in response to β-(1,6)-glucan. This study establishes a class of common pathogens as functional ligands for a subset of somatically mutated human B cell lymphomas.

A Pilot Phase II Study of the Lyn Kinase Inhibitor Bafetinib in Patients with Relapsed or Refractory B Cell Chronic Lymphocytic Leukemia

Abstract▪2858▪This icon denotes a clinically relevant abstractInhibition of B cell receptor signaling is an important mechanism of controlling the proliferation and survival of B cell chronic lymphocytic leukemia (CLL) cells. Lyn, a member of the Src kinase family, couples the B cell receptor to downstream signaling and is over expressed in CLL cells relative to normal B lymphocytes. Lyn is upstream from regulatory kinases in B cells such as PI3 kinase Δ and Bruton’s tyrosine kinase, which have been successfully targeted for the treatment of CLL. Addition of Lyn kinase inhibitors to CLL cell cultures accelerates apoptosis; treatment of CLL cells with drugs that induce apoptosis decreases both the activity and amount of Lyn kinase (Contri et al, J Clin Invest, 2005; 115 :369–78). These findings support a critical role for Lyn kinase in CLL pathogenesis by showing correlation between high basal Lyn activity and defects in induction of apoptosis in CLL cells. Thus bafetinib, a limited Src family kinase inhibitor targeting BCR/ABL, Lyn, and Fyn kinases, may suppress the growth of CLL cells by inhibiting Lyn kinase and thus have clinical activity. Methods and Objectives:We conducted a single arm, open-label phase 2 trial of single-agent bafetinib in patients with CLL who were relapsed or refractory to > 1course of treatment with either an alkylating agent, rituximab, or a fludaribine-based regimen. Patients were treated with oral bafetinib twice daily (BID), continuously until disease progression or unacceptable toxicity. Endpoints included response assessment by International Working Group CLL Criteria (Hallek et al, Blood, 2008) and toxicity. Results:At the time of analysis, 16 patients were administered bafetinib in the intent-to-treat population at 2 study sites (MD Anderson Cancer Center and City of Hope Cancer Center). The median age was 71 years (range 55–88 years); 13 patients were male. 13 patients were Caucasian and 3 were African-American. Of the 12 patients who were tested, 9 had del (17p;13). Patients had received a median of 3 prior treatment regimens (range 1–6). Where indicated, 5/16 (31%) patients were Rai stage I, 5/16 (31%) Rai stage II, 1/16 (6%) Rai stage III and 5/16 (31%) Rai stage IV. 14/16 patients had ECOG performance scores of 0 or 1. The median duration of treatment with bafetinib was 2 months (range 0.25 to 5 months). 11/16 patients were evaluable for response (baseline + > 1 post-baseline evaluation). There were no objective responses by IWCLL 2008 criteria. Partial nodal responses (> 50% reduction in bidimensional measurements in lymph nodes and/or > 50% shrinkage in spleen size) were observed in 7/11 evaluable patients. 2 patients had stable disease post baseline assessments and 2 patients demonstrated progressive disease at their initial evaluation post baseline. 14 patients were evaluable for toxicity. Almost all of the treatment-related adverse events were grade 1 or 2, with fatigue, nausea and elevated liver enzymes being the most common. Grade 1–2 elevated liver enzymes occurred in 9/14 patients; 1/14 patients had a grade 3 elevated liver enzymes and was removed from the study. No patients exhibited elevated bilirubin. One patient with Parkinson’s disease had a worsening of his symptoms and was withdrawn from the trial. No treatment-related serious adverse events occurred. Conclusion:Bafetinib, a Lyn kinase inhibitor, shows promise in patients with relapsed/refractory B-CLL at a dose of 240 mg orally BID. No treatment-limiting toxicity was observed at this dose level. Preliminary PK analysis from a parallel solid tumor study indicated that peak bafetinib concentrations at this dose only reach 0.3–0.6 μM, which is below the concentrations that inhibit the growth of B-CLL cells in vitro (1–1.5 μM). A higher dose level of 360 mg orally BID is being investigated. Future studies involving combinations of bafetinib with monoclonal antibodies and chemotherapy are planned. Disclosures:Kadia:CytRx Corporation: Research Funding. Delioukina:CytRx Corporation: Research Funding. Wieland:CytRx Corporation: Employment, Equity Ownership. Levitt:CytRx Corporation: Employment, Equity Ownership.

SET oncoprotein overexpression in B-cell chronic lymphocytic leukemia and non-Hodgkin lymphoma: a predictor of aggressive disease and a new treatment target

AbstractB-cell chronic lymphocytic leukemia (CLL), an incurable leukemia, is characterized by defective apoptosis. We found that the SET oncoprotein, a potent inhibitor of the protein phosphatase 2A (PP2A) tumor suppressor, is overexpressed in primary CLL cells and B-cell non-Hodgkin lymphoma (NHL) cell line cells. In CLL, increased levels of SET correlated significantly with disease severity (shorter time to treatment and overall survival). We developed SET antagonist peptides that bound SET, increased cellular PP2A activity, decreased Mcl-1 expression, and displayed selective cytotoxicity for CLL and NHL cells in vitro. In addition, shRNA for SET was cytotoxic for NHL cells in vitro. The SET antagonist peptide COG449 inhibited growth of NHL tumor xenografts in mice. These data demonstrate that SET is a new treatment target in B-cell malignancies and that SET antagonists represent novel agents for treatment of CLL and NHL.

CD160 signaling mediates PI3K-dependent survival and growth signals in chronic lymphocytic leukemia

AbstractB-cell chronic lymphocytic leukemia (CLL) expresses CD160, a glycosylphosphatidylinositol-linked receptor found on normal natural killer (NK) and T cells, but not B cells. CD160 is a multifunctional molecule in normal lymphocytes, but its role in CLL biology is unknown. In vitro, CLL cells undergo rapid spontaneous apoptosis, which CD160 activation protected against—mean cell viability increased from 67% to 79% (P < .001). This was associated with up-regulation of Bcl-2, Bcl-xL, and Mcl-1, but not Bax. As expected from these changes in Bcl-2/Bax and Bcl-xL/Bax ratios, CD160 triggering reduced mitochondrial membrane potential collapse and cytochrome c release. CD160 stimulation also induced DNA synthesis, cell cycle progression, and proliferation. B-cell antigen receptor (BCR)–induced CLL proliferation was generally greater than with CD160, but marked variation was seen. Both BCR and CD160 signaling led to CLL secretion of interleukin-6 (IL-6) and IL-8, although CD160 induced greater increases of IL-6 (51-fold) and IL-8 (15-fold). Survival and activation signals mediated by CD160 showed dose-dependent suppression by phosphoinositide-3 kinase (PI3K) inhibitors. Thus, in vitro, CLL cells can use the CD160 pathway for survival and activation, mimicking CD160 signaling in normal NK and CD8+ T cells. Establishing the pathophysiologic relevance of these findings may reveal new therapeutic targets.

Spleen Tyrosine Kinase Inhibition Prevents Chemokine- and Integrin-Mediated Stromal Protective Effects in Chronic Lymphocytic Leukemia.

Abstract 2356Poster Board II-333B cell chronic lymphocytic leukemia (CLL) remains incurable despite recent therapeutic advances, and identification of novel molecular targets in CLL is highly desirable. The microenvironment provides essential growth and survival signals to CLL cells and confers resistance to cytotoxic agents, a phenomenon termed cell adhesion-mediated drug resistance (CAM-DR). The chemokine CXCL12 and the integrin ligand VCAM have been identified as important microenvironmental pathways that mediate CLL cell survival and CAM-DR. We have previously demonstrated that inhibition of spleen tyrosine kinase (SYK), a key component of the BCR signaling pathway, induces apoptosis in primary CLL cells when cultured alone or supported by stroma (Buchner et al., Cancer Res. 2009). In this study, we investigated SYK inhibition with regard to the interaction between CLL cells and the microenvironment.Stromal cell coculture resulted in SYK activation in primary CLL cells as indicated by phospho-protein analysis by flow cytometry (p<0.001) and immunoblotting. In vitro stimulation with 20 ng/ml recombinant CXCL12 or VLA-4 integrin ligation by contact to VCAM-1 precoated wells (10 μg/ml) for 15 sec had equivalent effects. SYK inhibition by the pharmacological SYK inhibitor R406 significantly reduced F-actin polymerization and Akt phosphorylation induced by CXCL12, integrin ligand, or stromal contact. In functional experiments, migration of CLL cells towards CXCL12 was reduced to 77% (p<0.001) by R406 in transwell culture. Likewise, CLL cell adhesion to VCAM was limited by SYK inhibition to 50% (p<0.01) compared to control cells in flow chamber experiments. In response to a number of physiological stimuli, including BCR ligation, integrins are activated through the modulation of their conformation to mediate high-affinity adhesion. Analysis of the expression of a high affinity epitope of CD29, the β1 chain of VLA-4, on CLL cells by flow cytometry with and without SYK inhibition revealed a significant decrease of 31% (p<0.0001), while the expression of total CD29 decreased only by 10% (p<0.01). To verify the specificity of the effects of pharmacological SYK inhibitors, we performed siRNA mediated silencing of SYK in primary CLL cells. 48 h after transfection, we observed a downregulation of SYK protein expression by flow cytometry by 27% (p<0.05), while the viability of the cells was not significantly affected for 72 h. Stimulation of control- and SYK-siRNA treated cells with VCAM significantly increased Akt phosphorylation in control but not in SYK-siRNA transfected cells. Furthermore, SYK-silenced CLL cells adhered significantly less to VCAM-coated surfaces than control-silenced CLL cells.Beyond inhibition of these defined pathways, R406 abrogated the protective effect of stromal coculture on CLL cell survival. The anti-apoptotic factor Mcl-1 was upregulated in CLL cells cultured with stroma compared to those cultured alone (mean MFI ± SEM: 144 ± 21 vs. 182 ± 23, p<0.001). SYK inhibition almost completely abolished the upregulation of Mcl-1 in stroma-cocultured CLL cells (mean MFI ± SEM: 182 ± 23 vs. 130 ± 15, p<0.001). Finally, SYK inhibition also prevented CAM-DR against fludarabine in stroma coculture.Taken together, our data demonstrate direct effects of SYK inhibition on chemokine and integrin-mediated CLL-stroma interactions and establish a novel therapeutic mechanism of action for SYK inhibitors in CLL. Indeed, a promising clinical activity in patients with relapsed/refractory CLL/SLL with an overall response rate of 54% has been reported for the oral prodrug of R406, fostamatinib disodium in CLL patients (Friedmann et al, ASH 2008). Of note, an unexplained transient but dramatic leukocyte flare during lymph node regression was reported in several of these patients. Our results offer a convincing explanation for this phenomenon and may even suggest that SYK blockade in CLL is a promising therapeutic principle not only due to inhibition of BCR signaling, but perhaps even more importantly by disrupting the contact of CLL cells with their protective microenvironment. Disclosures:No relevant conflicts of interest to declare.

Targeting CK2 Overexpression as a Novel Therapeutic Tool in Chronic Lymphocytic Leukemia.

Abstract 803Characterization of the molecular mechanisms that regulate viability of B cell chronic lymphocytic leukemia (CLL) cells may provide novel insights into the biology of this incurable malignancy and reveal prognostic markers and therapeutic targets. In particular, specific inhibition of signaling elements essential for leukemia cell survival offers great promise for the design of more efficient and selective therapies. The protein serine/threonine kinase CK2 is frequently upregulated in different cancers and mounting evidence implicates CK2 in tumorigenesis. In the present study, we evaluated whether CK2 may play a significant role in CLL. Peripheral blood mononuclear cells (PBMCs) from CLL patients (n=15) and healthy controls (n=7 ) were initially compared by Western blot densitometry analysis. CK2α, but not CK2β, was significantly upregulated in primary CLL patient samples (P=0.0003; Unpaired t-test), and CK2α expression clearly correlated with CK2 in vitro kinase activity (P value=0.0165). To test the functional significance of CK2 overexpression in CLL, we cultured PBMCs collected from leukemia patients (n=53) or purified CLL cells (n=7) in the presence of the CK2-specific small molecule inhibitors TBB and DRB. As determined by Annexin V-APC/7AAD staining and analysis of procaspase 3 cleavage, both TBB and DRB clearly promoted apoptosis of CLL cells (CD19+CD5+) in a time- and dose-dependent manner. Importantly, neither CK2 antagonist induced significant cell death in the normal T-cell population (CD19-CD3+) present in each PBMC patient sample. Consequently, the percentage of normal T-cells dramatically increased upon TBB/DRB treatment. Further, B-cells from healthy controls (n=7) were not affected by TBB or DRB, confirming the selectivity of the CK2 inhibitors towards leukemia cells. Notably, although co-culture with OP9 stromal cells promoted primary leukemia cell survival in vitro, it did not prevent apoptosis of CLL cells mediated by CK2 inhibition. The pro-apoptotic effect of TBB/DRB did not correlate with clinical parameters such as lymphocyte doubling time, ZAP-70 expression or IGHV mutational status. Interestingly, there was a significant association between the percentage of CLL cells in the peripheral blood and sensitivity to CK2 inhibition (e.g. 25μM TBB, P=0.0043), and treatment with 12.5μM TBB correlated with Binet stage (P=0.0043). We previously showed that CK2 phosphorylates and thereby inactivates PTEN in primary T-ALL cells leading to the hyperactivation of PI3K signaling pathway (Silva et al, JCI 2008). Here, we found that primary CLL samples displayed higher P-PTEN/PTEN ratios than healthy controls (P=0.0034), which correlated with CK2 expression (P=0.0064). Furthermore, treatment with TBB/DRB abrogated PTEN phosphorylation in 2 primary leukemia samples analyzed, suggesting that CK2 negatively regulates the activity of the tumor suppressor PTEN in CLL and raising the possibility that the pro-survival effect of CK2 may be dependent, at least in part, on the activation of PI3K pathway. Overall, our study suggests that CK2 plays an important role in the biology of CLL, and lays the groundwork for the inclusion of CK2 antagonists into future therapeutic options. Disclosures:No relevant conflicts of interest to declare.

B-Cell Chronic Lymphocytic Leukemia Cells Exposed to the Non-Genotoxic p53 Activator Nutlin-3 Are Characterized by a Specific Gene Expression Signature.

Abstract 4374 Introductionp53 plays a key role in determining the clinical features of B cell chronic lymphocytic leukemia (CLL). Disruption of p53 by point mutations, deletion at 17p13, or both, occurs in a fraction of cases at diagnosis and predicts poor survival and chemorefractoriness. In cells with functional p53, p53 activity is inhibited through interaction with MDM2. In fact, p53 can be activated upon exposure of cells to inhibitors of p53/MDM2 interaction, like Nutlins. Exposure of CLL cells to Nutlin-3 is effective in raising the levels of p53 protein with subsequent induction of cell cycle arrest and/or apoptosis, independently of the most relevant prognostic markers. The aim of the present study was to analyze the gene expression profile (GEP) induced by Nutlin-3 exposure in primary CLL cells from p53wt and p53del/mut cases. Patients and methodspurified cells from 24 PB CLL samples, all characterized for IGHV mutational status, CD38 and ZAP-70 and p53 mutations (16 p53wt CLL, 8 p53del/mut CLL of which 6 with del17p13 and p53 mutations, 1 with del17p13 alone, and 1 with p53 mutations alone), were exposed to 10 mM Nutlin-3 for 24 hours. GEP was performed using a dual labelling strategy; the differential expression of the below reported genes were validated by quantitative real-time PCR. Resultsi) signature of Nutlin-3 exposure in p53wt CLL: 144 differentially expressed genes (143 up-regulated, 1 down-regulated) were correlated with response to Nutlin-3. Among the over-expressed genes, several genes were related to apoptosis (e.g. BAX, BBC3, E124, IKIP, FAS, LRDD, FLJ11259, TRIAP1, GADD45, TP53INP1, ISG20L1, ZMAT3, TNFRS10C, TNFRSF10B/TRAIL-R2), while other genes (e.g. MDM2, CDKN1A, PCNA) were up-regulated by Nutlin-3 as a part of a negative feed-back mechanism. Of note, this signature was not shared by 3/16 p53wt cases (identified as “non-responder” p53wt CLL) and 7/8 p53del/mut cases (identified as “non-responder” p53del/mut CLL); consistently, cells from these cases were also significantly resistant to the in-vitro cytotoxic effects of Nutlin-3; ii) signature of Nutlin-3 “non-responder” p53wt CLL: by comparing the constitutive GEP of 13 “responder” versus 3 “non-responder” p53wt CLL, we obtained 278 differentially expressed genes, 149 up-regulated and 129 down-regulated in “non-responder” p53wt CLL. Among up-regulated genes, we focused on MDM4/MDMX, a gene whose product was known to have an inhibitor activity of p53-dependent transcription and to form Nutlin-3 resistant complexes with p53. Among down-regulated genes, validations were made for BIRC4BP, whose product is known to act as an antagonist of the anti-apoptotic protein XIAP; iii) signature of Nutlin-3 “non-responder” p53del/mut CLL: by comparing the constitutive GEP of 13 “responder” versus 7 “non-responder” p53del/mut cases, we obtained 72 differentially expressed genes, 26 up-regulated and 46 down-regulated (31/46 located at the 17p segment) in “non-responder” p53del/mut CLL. Validations were made for several genes whose products display pro-apoptotic activities (e.g. PSMB6, RPL26 and ZBTB4, located at 17p segment, and GNAZ located at chromosome 22) among down-regulated genes, and ARHGDIA, whose gene product displays anti-apoptotic activities and mediates cellular resistance to chemotherapeutic agents, among up-regulated genes. Notably, CLL cells (n=43) displayed constitutively higher levels of MDM4/MDMX (p<0.0001) and ARHGDIA (p=0.0002) transcripts than purified normal B cells (n=15), irrespectively to the major biologic prognosticators. Conclusionsspecific gene-sets and GEP were documented to be associated with response or resistance to Nutlin-3 exposure in p53wt or p53del/mut CLL. These findings may help to identify novel molecular targets for CLL therapy. Disclosures:No relevant conflicts of interest to declare.

Targeted knockdown of the B lineage transcriptional activator E2A in B cell chronic lymphocytic leukemia (CLL) enhances lenalidomide sensitivity and regulates cellular proliferation (88.7)

Abstract Chronic lymphocytic leukemia (CLL) is a common malignancy characterized by the accumulation of B lymphocytes with an antigen-experienced, activated CD19+CD5+ clonal phenotype. Clinically, approximately 50% of cases will behave more aggressively. Here we used siRNA knockdown to investigate the contribution of E2A, a key transcriptional regulator of B cell activation, to CLL survival and proliferation. E2A silencing in primary CLL cells leads to a modest increase in spontaneous apoptosis in both CD38+ (aggressive) and CD38- (indolent) cases. E2A is known to restrain lymphocyte proliferation, and E2A disruption promotes cell cycle progression of aggressive but not indolent CLL. Lenalidomide, an immunomodulatory (IMiD) anticancer drug with pro-apoptotic activity in some B lineage cancers, shows synergistic effects with E2A knockdown. While lenalidomide alone fails to induce detectable apoptosis in CLL, the combined treatment of lenalidomide plus E2A silencing dramatically increases apoptosis beyond either treatment alone. Thus, disruption of a transcription factor associated with the B cell activation profile enhances the anti-tumor potential of lenalidomide, providing the opportunity for improved drug efficacy.

Spleen Tyrosine Kinase (SYK) Is Overexpressed and Represents a Potential Therapeutic Target in Chronic Lymphocytic Leukemia

B cell chronic lymphocytic leukemia (CLL), the most prevalent B cell malignancy in adults, is characterized by expansion of monoclonal mature B lymphocytes. Despite advances in treatment, the disease remains incurable warranting further efforts to identify novel molecular targets in CLL. B cell receptor (BCR) signaling contributes to apoptosis resistance in CLL limiting the efficacy of therapeutic approaches. In this study we investigated the expression of spleen tyrosine kinase (SYK), a key component of the BCR signaling pathway, in CLL and its role in apoptosis. Gene expression profiling identified enhanced expression of SYK and downstream pathways in CLL compared to healthy B cells. Immunoblotting showed increased expression and phosphorylation of SYK, PLCγ2, STAT3, and ERK1/2 in CLL compared to healthy B cells suggesting enhanced activation of these mediators in CLL. Separate analyses according to prognostic parameters revealed 1.8-fold higher SYK protein level in unmutated compared to mutated CLL cells determined by densitometric analysis (n=22, p=0.0031). These findings may well explain the higher BCR signaling capacity in the unmutated CLL subset. Various SYK inhibitors (piceatannol, curcumin, SYK Inhibitor II, and IV (Calbiochem)) reduced phosphorylation of the SYK downstream targets PLCγ2, STAT3, and ERK1/2 in a time- and dose-dependent manner and induced apoptosis in the CLL cell lines Mec-1 and EHEB and primary CLL cells. SYK Inhibitor II revealed highest cytotoxic effects on primary CLL cells, but did not significantly reduce the viability of healthy B cells. Thus, apoptotic effects of this inhibitor were analyzed in a larger cohort of patient samples along with the well-established SYK inhibitor R406 (Rigel Inc.). After 48 hour treatment, relative viability of CLL cells was reduced to 76% and 44% for SYK Inhibitor II (4 mM and 10 mM) and to 66% for R406 (4 mM), respectively (n=38, p<0.0001). With respect to prognostic factors, SYK inhibitors exerted stronger cytotoxic effects in unmutated and ZAP70+ cases. Cytotoxicity of SYK inhibitors were associated with SYK protein expression potentially predicting response to therapy (Pearson correlation coefficient: r=0.78 for SYK Inhibitor II (p<0.0001) and r=0.56 for R406 (p=0.0134)). Combination of fludarabine with SYK Inhibitor II or R406 increased cytotoxicity compared to fludarabine therapy alone. The mean viability of F-ara-A treated cells (10 mM) was reduced by 13% with SYK II and by 17% with R406 (n=10, p<0.0001), respectively. Since microenvironment enhances the viability of CLL cells and decreases their sensitivity towards chemotherapy, apoptosis rates of CLL cells incubated with SYK Inhibitor II in the presence and absence of the stromal cell line M2-10B4 were assayed. No significant change in cytotoxic effects was observed. Hence, stromal cell interactions do not impair the cytotoxicity of SYK inhibitors. Moreover, CD40L expressing T cells play an important role in the CLL microenvironment, and CD40 ligation is under discussion to induce fludarabine resistance in CLL. Therefore the effect of SYK Inhibitor II in combination with CD40 ligation was analyzed. In contrast to conventional chemotherapy, stimulation with CD40L significantly sensitized CLL cells towards SYK inhibition. In conclusion, this work establishes SYK inhibition as a rational and promising therapeutic principle in CLL. Given the preferential activity of SYK inhibitors in CLL cases with poor prognostic factors, the independence of their activity from the protective influence of the CLL microenvironment, and the synergistic and complementary action with fludarabine, we propose SYK inhibitors for clinical assessment in the therapy of CLL.

Cytotoxicity of the Type 4 Phosphodiesterase Inhibitor CD160130 for Freshly Isolated Human CLL Cells In Vitro.

B cell chronic lymphocytic leukemia (CLL), the most common subtype of leukemia in the United States of America and in Europe, is treatable but incurable. New drugs are needed for its management. Phosphodiesterase inhibitors (PDEi) block catabolism of cyclic nucleotides resulting in accumulation of cellular cAMP and cGMP. These PDEi also decrease production of inflammatory cytokines such as tumor necrosis factor. Furthermore, they inhibit expression of inducible nitric oxide synthase (NOS2) mRNA and NO production. Researchers have previously noted that nonspecific PDE inhibitors such as theophylline as well as a relatively specific PDE4 inhibitor (rolipram) cause CLL cell death in vitro, while relatively sparing normal peripheral blood mononuclear cells (PBMC). The purpose of this study was to evaluate the effectiveness of CD160130 in the killing of freshly isolated CLL cells in vitro. CD160130 is a novel, orally available heterocyclic pyrimido-indole derivative that is relatively PDE4-specific. Patients were from the V.A. and Duke University Medical Centers, and normal controls were from the community. Control normal PBMC were isolated by ficoll-Hypaque centrifugation, and CLL cells were purified using negative selection with antibodies. We determined cytotoxicity using the MTS colorimetric assay. Samples from 10 CLL patients (6 male and 4 female) and 10 normal controls were examined. Nine of 10 patients were stage 0 at presentation, and 1 was stage 2. They had been followed 6.1 yr (median; range 0.7–19.1 yr). One of 10 was CD38 positive, and 6 of 10 were Zap-70 positive. Of nine analyzed, one had unmutated IgVH gene, and 9 were mutated. Six patients had not been treated, and 4 had been treated with chlorambucil, fludarabine, and/or rituximab. Four had normal cytogenetics by FISH analysis; one had trisomy 12; three had 13q14 del; and one had 17p del. Of seven determined, two had elevated CLL cell lipoprotein lipase mRNA elevated. CD160130 induced cell death in a dose-dependent fashion in all patients' samples, with a mean cytotoxicity of 96% at 12.5 uM. The mean ED50 for killing was 233 nM in media with FBS and 314 nM in serum-free medium, while that for PBMC was higher at 7500 nM with FBS and 5210 nM with serum-free medium. The agent was 32.2 fold more potent for killing of CLL cells compared to PBMC in medium with FBS and 16.6 fold more potent for CLL cells in serum-free medium. In summary, the PDE4 inhibitor CD160130 potently kills freshly isolated CLL cells in vitro in the presence or absence of serum. The killing is relatively selective for CLL cells compared to normal PBMC. In vivo trials of CD160130 in patients with CLL should help determine the toxicity and efficacy in patients.