A platform for phenotypic discovery of therapeutic antibodies and targets applied on Chronic Lymphocytic Leukemia

VSV Oncolysis in Combination With the BCL-2 Inhibitor Obatoclax Overcomes Apoptosis Resistance in Chronic Lymphocytic Leukemia

Murine TCL1 CLL Cells with B-Cell Receptors Specific for the Autoantigen Phosphatidylcholine Have a Selective Advantage During Adoptive Transfer

Matriptase Is Highly up-Regulated in Chronic Lymphocytic Leukemia and Promotes Cancer Cell Invasion

Novel Oncogenic Non-Coding RNA：circRIC8B Regulates Lipid Metabolism Via Mir-199b-5p /LPL Axis in Chronic Lymphocytic Leukemia

Tumor Heterogeneity-Based Sscr-Score Predicts Prognosis and Reveals Mechanism of SORL1 in Restraining CLL Progression

The Dual PI3K/mTOR Inhibitor PF-04691502 Induces Substantial Apoptosis in Chronic Lymphocytic Leukemia Cells in Vitro and Prolongs Survival in the Eµ-TCL1 Mouse Model

Recently, systemic administration of a human monoclonal antibody directed against cytotoxic T lymphocyte-associated antigen 4 (CTLA-4) expressed on circulating T cells in patients with chronic lymphocytic leukaemia (CLL) has been considered. Also, CLL cells have been shown to express CTLA-4, increased levels of which in the leukaemic compartment are a predictor of good clinical outcome. Since both CLL and Treg microenvironment cells can be targeted by the CTLA-4 blocking antibody in this immunotherapy approach, the investigation of the functional effect of CTLA-4 blockade on CLL cells might be of potential clinical relevance. The main aim of this study was to examine the effect of CTLA-4 blockade on proliferation activity and apoptosis of CLL cells in patients with low and high CTLA-4 expression. We found that in the high CTLA-4-expressing CLL group, CTLA-4 blockade on the CLL cell surface resulted in a significant increase in the median percentages of Ki67+ cells and a tendency to decrease in the proportion of apoptotic cells. In contrast, in the low CTLA-4 expressors, CTLA-4 blockade did not affect the proliferation activity or the frequency of apoptosis. This study reports for the first time the different effect of CTLA-4 blockade on CLL cells in CLL patients depending on the levels of CTLA-4 expression. CTLA-4 blockade seems to induce pro-survival signals in leukaemic cells from CLL patients exhibiting high CTLA-4 expression, suggesting that an immunotherapy approach based on the systemic use of monoclonal anti-CTLA-4 antibodies could be an unfavourable strategy for some CLL patients.

<div>AbstractPurpose:<p>Although the B-cell receptor (BCR) signal plays a critical role in chronic lymphocytic leukemia (CLL) cell survival and a target of current therapies (ibrutinib targets Bruton’s tyrosine kinase; idelalisib targets PI3Kδ), contribution of the cytokine-driven JAK2 pathway to the “CLL cell-survival signaling network” is largely undefined.</p>Experimental Design:<p>Patients with CLL were enrolled to investigate expression/activation of JAK2 and acylglycerol kinase (AGK), and their functional implication in primary CLL cell survival. A series of biochemical and molecular biology assays were employed to uncover the underlying mechanism.</p>Results:<p>We detected that compared with normal B cells, CLL cells aberrantly express constitutively active JAK2. Mechanistically, HSP90 forms a chaperoning complex with JAK2, resulting in its aberrant accumulation in CLL cells. We also discovered aberrant upregulation of a novel mitochondrial lipid kinase, AGK, which remains complexed with HSP90 in CLL cells activating JAK2. Although AGK is typically mitochondrial, we detected its nuclear localization in association with JAK2 in some CLL cells. Functionally, JAK2 phosphorylates its noncanonical substrate, histone H3(Y41), but not STAT3, activating transcription of diverse sets of genes in a patient-specific manner. Additionally, JAK2 activates the BCR signal in CLL cells via LYN/Bruton’s tyrosine kinase axis. Targeted inhibition of JAK2 as monotherapy, or in combination with the BCR inhibitors or venetoclax (a BCL2 inhibitor), induced apoptosis synergistically in CLL cells.</p>Conclusions:<p>These findings suggest that aberrantly expressed AGK activates JAK2, independent of cytokine, leading to activation of diverse sets of gene transcription in CLL cells. Combined targeting of JAK2 and BCR signals or BCL2 may be effective in some patients with CLL.</p></div>

Introduction: Regulatory B cells (B-reg cells) and cytokine producing B cells have been increasingly recognized as key regulators of adaptive and innate immunity. Malignant B cells, such as chronic lymphocytic leukemia (CLL) cells share many features of B-reg cells and suppress host adaptive immunity via multiple mechanisms, including production of suppressive cytokines such as IL-10, surface expression of checkpoint molecules such as PD-L1 and CD200, as well as inducing expansion of regulatory T cells and Myeloid-derived Suppressor Cells (MDSC). Ibrutinib is an orally available irreversible BTK inhibitor that has been proved to be a safe and effective therapy for CLL. Recently we and others have demonstrated favorable cellular immunomodulatory effects of Ibrutinib through inhibition of an alternative target interleukin-2 induced T-cell kinase (ITK) that promotes Th1/Tc1 polarization. Herein, we explore the influence of Ibrutinib on the immune suppressive features and the B-reg phenotype associated with malignant B cells. Methods: PBMCs were collected from CLL patients treated with Ibrutinib at the time of pretreatment, cycle 3 day 1 (8 weeks into treatment) and cycle 6 day 1(20 weeks into treatment). Surface expression of checkpoint molecules and immunosuppressive capability of CLL cells were evaluated. For brief stimulation (“B10” condition), PBMCs were stimulated with Phorbol Myristate Acetate (PMA)/Ionomycin/Golgi-stop plus CpG for 5 hours. For prolonged stimulation (“B10-Pro” condition), PBMCs were stimulated with CpG plus CD40L for 48 hours, PMA / Ionomycin / Golgi-stop were added for final 5 hours. Results: CLL cells collected from patients treated with Ibrutinib in vivo were significantly impaired in their capability to make IL-10 after in vitro stimulation. On average, there is about a threefold reduction (P< 0.001) in the frequency of cells producing IL-10 by cycle 3, and a fivefold reduction (P< 0.001) by cycle 6. We have also shown that during the first two cycles of Ibrutinib, patients' plasma levels of IL-10 decreased. The impaired IL-10 production by CLL cells collected from post-Ibrutinib treatment patients is likely to due to an intrinsic change of the malignant B cells, rather than from acute inhibition of BTK; since resting these CLL cells in vitro for 48 hours (which allows for regeneration of BTK) did not lead to a recovery of IL-10 production. Interestingly, IFNγ production after in vitro re-stimulation is increased in CLL cells collected post Ibrutinib treatment. The analysis of potential immunosuppressive molecules revealed 3-5 fold reduction in surface expression of CD200 and BTLA(P<0.001 for both), but no significant change in the expression of PD-L1 or B7-H3 in CLL cells collected post Ibrutinib treatment compared to pre-treatment samples. Given that B-regs can influence both T-regs and MDSCs, we exammined these and found the percentage of CD4+/CD25+/Foxp3+ regulatory T cells and CD14+/HLADRlow Myeloid-derived Suppressor Cells were significantly reduced after Ibrutinib treatment(P<0.001 and P<0.05). Conclusion: CLL cells cause both global and tumor specific immunosuppression via multiple mechanism recapitulating regulatory B cells including production of IL-10, expression of checkpoint molecules such as CD200, and induction of suppressive/regulatory cells such as Treg cells and MDSC cells. Here we demonstrate that Ibrutinib treatment has a powerful in vivo immunomodulatory effect on the malignant B cells by counteracting all the aforementioned B-reg like activities, which makes it a promising immunotherapeutic agent. Note: This abstract was not presented at the conference. Citation Format: Meixiao Long, Kyle A. Beckwith, Priscilla Do, Amber Gordon, Amy Lehman, Kami Maddocks, Carolyn Cheney, Jeffrey Jones, Leslie Andritsos, Farrukh Awan, Jennifer Woyach, Johnson J. Amy, Natarajan Muthusamy, John Byrd. Ibrutinib treatment counteracts the immunosuppressive activity of malignant B cells [abstract]. In: Proceedings of the Second CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; 2016 Sept 25-28; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(11 Suppl):Abstract nr B041.

Clinical utility of assessing ZAP‐70 and CD38 in chronic lymphocytic leukemia

Bruton's Tyrosine Kinase Inhibitor PCI-32765 Abrogates BCR- and Nurselike Cell-Derived Activation of CLL Cells In Vitro and In Vivo.

In Vivo Effects of Ibrutinib On BCR Signaling, Tumor Cell Activation and Proliferation in Blood and Tissue-Resident Cells of Chronic Lymphocytic Leukemia Patients

The Role of Proteasome Inhibitors and the Trail Apoptotic Pathway in the Treatment of Chronic Lymphocytic Leukemia.

Introduction: Escape from immunosurveillance is a hallmark of chronic lymphocytic leukemia (CLL) cells. In the protective niche of lymphoid organs, leukemic cells suppress the ability of T lymphocytes to form the immune synapse (IS), thereby hampering T-cell mediated anti-tumoral activities. By binding its cognate receptor PD-1 at the surface of T lymphocytes, the inhibitory ligand PD-L1, which is overexpressed in CLL cells, mediates the T-cell suppressive activities of CLL cells. However, the molecular mechanism underlying PD-L1 overexpression in CLL cells remains unknown. We have previously reported a defective expression of the pro-apoptotic and pro-oxidant adaptor p66Shc in CLL cells, which is causally related to an impairment in intracellular reactive oxygen species (ROS) production and to the activation of the ROS-sensitive transcription factor NF-κB. The fact that PD-L1 expression is regulated by NF-κB suggests a mechanistic relationship between p66Shc deficiency and PD-L1 overexpression in CLL cells. Methods: 62 treatment-naive CLL patients and 43 healthy donors were included in this study. PD-L1 and p66Shc expression was quantified in B cells by flow cytometry and qRT-PCR. IS architecture and local signaling was assessed by flow cytometry and confocal microscopy. CD8+ cell killing activity was assessed by flow cytometry. Results: Here we show that residual p66Shc expression in leukemic cells isolated both from CLL patients and from the CLL mouse model Eμ-TCL1 inversely correlated with PD-L1 expression. We also show that the PD-L1 increase prevented leukemic cells from forming ISs with T lymphocytes. Reconstitution of p66Shc, but not of a ROS-defective mutant, in both CLL cells and the CLL-derived cell line MEC-1, enhanced intracellular ROS and decreased PD-L1 expression. Similar results were obtained following treatment of CLL cells with H2O2 as exogenous source of ROS, that normalized PD-L1 expression and recovered IS formation. Discussion: Our data provide direct evidence that the p66Shc-deficiency-related ROS depletion in CLL cells concurs to enhance PD-L1 expression and provides a mechanistic basis for the suppression of T cell-mediated anti-tumoral functions in the immunosuppressive lymphoid niche.

Cytokine and T-Cell Phenotypic Changes Upon In Vivo Ibrutinib Therapy For CLL – Targeting Both CLL Cells and The Tumor-Microenvironment