Cells Of CLL Patients Research Articles

IL-25 Impact on Malignant B Cells Survival and T Cells Activation in Chronic Lymphocytic Leukemia.

T cell dysregulation and shift to T helper 2 responses, boosting tumor microenvironment support, contributes to the survival of leukemic B cells in Chronic Lymphocytic Leukemia. Interleukin (IL)-25 is involved in the initiation of T helper 2 cell responses. Signal transduction of IL-25 begins with the heterodimer receptor (IL-17RA/IL-17RB). The presence of IL-25 in the tumor microenvironment may affect the supportive effects of T cells in the surrounding tumor cell environment. The purpose of this study was to evaluate the role of IL-25 in the biology of CLL. IL-17RB expression in CD3+ and CD19+ cells was assessed in isolated peripheral blood mononuclear cells (PBMCs) of nine CLL patients and nine healthy subjects by real-time polymerase chain reaction and flow cytometry. B cells were positively enriched from PBMCs using magnetic-activated cell sorting (MACS). PBMCs and purified leukemic B cells were cultured with recombinant human IL-25 (20ng/ml) for 72 hours, then the viability and apoptosis of cultured cells were measured by MTT assay and AnnexinV/7AAD. Furthermore, the levels of CD69 expression on T lymphocytes and IL-17RB in T and B cells were determined by flow cytometry. The basal level of IL-17RB expression in CLL patients was significantly higher than that in control individuals. In addition, the percentage of IL-17RB+/CD3+, IL-17RB+/CD19+ cells and CD69+/CD3+ cells increased after 72 hours of culture with IL-25 in CLL patients compared to healthy subjects. IL-25 also reduces the apoptosis rate of tumor cells. We found that IL-25 could stimulate T cells in CLL patients and lower B cell death. This suggests that IL-25 might have a role in enhancing the survival of tumor cell by expressing receptors for inflammation, such as IL-17RB, and might be involved in the development of CLL.

Elevated levels of circulatory follicular T helper cells in chronic lymphocytic leukemia contribute to B cell expansion.

Chronic lymphocytic leukemia (CLL) is characterized by an expansion of mature B cells in the bone marrow, peripheral lymphoid organs, and blood. CD4 T helper (Th) lymphocytes significantly contribute to the physiopathology of CLL, but the subset(s) of Th cell involved in CLL pathogenesis is (are) still under debate. In this study, we performed flow cytometry analysis of the circulatory T cells of untreated CLL patients and observed an increase in follicular helper T cells (Tfh), which is a subset of T cells specialized in B cell help. Elevated numbers of Tfh cells correlated with disease severity as measured by the Binet staging system. Tfh from CLL patients were activated and skewed toward a Th1 profile as evidenced by their PD-1+IL-21+IFNγ+ phenotype and their CXCR3+CCR6- chemokine receptor profile. Tfh efficiently enhanced B-CLL survival and proliferation through IL-21 but independently of IFNγ. Finally, we observed an inverse correlation between the Tfh1 and IgA and IgG serum levels in patients, suggesting a role for this Tfh subset in the immune dysfunction associated with CLL. Altogether, our data highlight an impairment in circulatory Tfh subsets in CLL patients and their critical role in CLL physiopathology.

Characterize Biomarkers and Mechanisms of Resistance for MDM2 Inhibitors in AML

Acute myeloid leukemia (AML) is a molecularly and clinically heterogeneous disease with an aggressive phenotype and poor prognosis. Inactivation of the wild-type p53, through upregulation of the TP53 negative regulator MDM2, is observed in around 50% of AML patients, providing a rationale for targeting MDM2 for the treatment of AML. Small molecule inhibitors of MDM2, including Idasanutlin, have shown efficacy as monotherapy and in combinations. However, patients exhibit heterogeneous responses with frequent drug resistance. We integrated ex vivo drug sensitivity assay, clinical parameters, whole exome sequence (WES), RNAseq, cytokine stimulation, and CRISPR/Cas9 dropout screening data to identify biomarkers that predict idasanutlin response and to elucidate the underlying drug-resistant mechanisms. Surprisingly, besides TP53 itself, we did not find an essential role in losing any of the major TP53 downstream targets alone leading to Idasanutlin resistance from Idasanutlin treated resistant cell lines, CRISPR screening, or individually targeted knockout experiments. Besides TP53 mutations, other major AML mutations, including RAS pathway mutations do not drive Idasanutlin resistance indicated by the patient sample functional screening data and in vitro mutation overexpression models. We observed instead that AML with a high differentiation or mature myeloid signature: including low blast, a high percentage of monocytes/granulocytes/T cells, AML FAB M4/M5 morphology, and a high expression of a myeloid immune signature gene are relatively resistant to Idasanutlin. We further characterized that both healthy/leukemia-associated T cells, granulocytes, and monocytes are intrinsically resistant to idasnutlin due to low expression levels of MDM2 and/or TP53. To examine whether the presence of differentiated cells protects leukemia blast cells from Idasanutlin, we performed a co-culture drug treatment experiment. Strikingly, only leukemia-associated monocytes, but not healthy monocytes, healthy/leukemia T cells or granulocytes, or leukemia blast cells protected leukemia blast cells from apoptosis in the presence or absence of Idasanutlin. Similar results were obtained for the other two MDM2 inhibitors, AMG232 and DS-3032b, and a BCL2 inhibitor, venetoclax. To further uncover which cytokines secreted from the leukemia-associated monocytes confer MDM2 inhibitor resistance, we tested 11 common cytokines, including IL-1a, IL-1b, IL-3, IL-6, IL-8, M-CSF GM-CSF, FLT3, MCP-1, TNFa, and SCF. We observed that IL-1α, IL-1β, IL-3, and GM-CSF markedly induced idasanutlin treatment resistance for AML, but not ALL or CLL patient cells. Interestingly, IL-1α and IL-1β only protect leukemia blasts, but not healthy CD34 hematopoietic stem and progenitor cells from Idasanutlin-induced apoptosis. We further performed RNAseq and immunoblots on IL-1α and IL-1β treated AML, CLL, and ALL cells. We observed the major TP53 downstream pathways remain activated by idasanutlin in the presence of IL-1α and IL-1β. Interestingly, GATA2 is significantly upregulated in IL-1α and IL-1β treated AML, but not CLL or ALL cells in the presence or absence of Idasanutlin. Furthermore, overexpressing GATA2 conferred Idasanutlin resistance at high concentrations. Finally, we observed that combining a FLT3/IRAK dual inhibitor, pacritinib, or an IRAK1 /IL-1 receptor antagonist, anakinra, partially rescued IL-1α and IL-1β mediated drug resistance of MDM2 inhibitors. As such, we uncovered the role of leukemia-associated monocyte in driving intrinsic and extrinsic MDM2 inhibitor resistance and the potential underlying mechanisms.

Abstract 3574: A Phase I trial of personalized multi-peptide vaccination in combination with the TLR1/2 ligand XS15 in CLL patients under Bruton-Tyrosin-Kinase-inhibitor based regimes - Preclinical development and study design

Abstract Chronic lymphocytic leukemia (CLL) is the most common leukemia in adults. Despite the therapeutic success of novel small molecules (e.g. ibrutinib, idealisib and venetoclax), patients frequently experience disease relapse due to the persistence of minimal residual disease (MRD). Targeting the residual CLL cells by immunotherapy could further improve the depth and persistence of CLL remission. Here, we report on the preclinical development and clinical implementation of a phase I trial evaluating safety, immunogenicity and efficacy in terms of MRD response of a personalized multi-peptide vaccine adjuvanted with the novel toll-like receptor (TLR) 1/2 agonist XS15 emulsified in Montanide࣪ ISA51 VG (Rammensee et al. JIC 2019) in CLL patient receiving Bruton-Tyrosin-Kinase (BTK)-inhibitor based regimes (NCT04688385). So far, broad application of peptide vaccines in cancer patients is hampered by time- and cost-intensive design and the lack of neoepitopes from tumor-specific mutations, especially in low-mutational burden malignancies. To improve on these drawbacks, we developed an immunopeptidome-guided workflow for the design of tumor-associated off-the-shelf peptide warehouses for broadly applicable personalized therapeutics (Nelde et al., Front Immunol, 2021). For the trial reported here, comparative mass spectrometry-based immunopeptidome analyses of 61 CLL patient samples and a dataset of benign tissues samples (n = 351) enabled the identification of high-frequent non-mutated CLL-associated antigens for the most common HLA allotypes (HLA-A*02, -A*24, and -B*07). These antigens were proven to be recognized as T cell epitopes by pre-existing and de novo induced T cells in CLL patients and healthy volunteers, thereby enabling the selection of a 12-peptides panel comprising 9 HLA-class I (3 for each HLA allotype) and 3 HLA class II restricted high frequent CLL-exclusive T cell epitopes as warehouse for the personalized composition of study vaccine cocktails. Our meanwhile ongoing phase I trial recruits CLL patients eligible for treatment with BTK inhibitors. For those with matching HLA allotypes, a personalized peptide-cocktail is formulated comprising 6 CLL-associated peptides (3 HLA class I and 3 HLA class II peptides), selected from our warehouse based on individual immunopeptidome analysis, and 2 highly immunogenic HLA class II control peptides, one viral (human adenovirus C) and one tumor associated (Baculoviral IAP repeat-containing protein 5). Three doses of the vaccine are administered in a 4-weekly interval, after reduction of CLL-load by BTK inhibitors. So far, 10 CLL patients were included in the trial. The first vaccinations will be applied in November and December 2021 with first immunogenicity data expected in March 2022 and to be reported at the meeting. Citation Format: Maddalena Marconato, Annika Nelde, Yacine Maringer, Marcel Wacker, Marion Richter, Monika Denk, Helmut Salih, Jonas Heitmann, Juliane Sarah Walz. A Phase I trial of personalized multi-peptide vaccination in combination with the TLR1/2 ligand XS15 in CLL patients under Bruton-Tyrosin-Kinase-inhibitor based regimes - Preclinical development and study design [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 3574.

Abstract 2102: NFAT2 determines susceptibility of chronic lymphocytic leukemia cells to constitutive and Rituximab-induced NK cell killing

Abstract Genetic loss of the transcription factor NFAT2 (NFATc1), known as important player in lymphocyte development, causes an aggressive course of CLL, as it enhances BCR signaling and leads to highly malignant and proliferative disease (Märklin et al. Nat Commun 2017). To investigate whether and how NFAT2 affects treatment resistance in CLL, we generated a CRISPR/Cas9 based NFAT2 knockout (KO) in MEC-1 CLL cells to model aggressive CLL. In immunodeficient NSG-mice, the NFAT2 KO cells showed a ten-fold higher engraftment in the bone marrow after three weeks compared to scrambled (SCR) controls. Cytotoxicity assays revealed a profoundly higher resistance of NFAT2 KO cells to NK cell killing without detectable differences at the level of recognition as revealed by analyses of NK cell activation, degranulation and IFNy release, which excluded resistance to NK cells was due to altered immunogenicity. As CLL patients are routinely treated with anti-CD20 antibody Rituximab, which mediates its therapeutic efficacy in large part by inducing antibody dependent cellular cytotoxicity (ADCC) of NK cells via secretion of perforin and granzymes. When we exposed the target cells to the cytotoxic content isolated out of NK cells, MEC-1 NFAT2 KO cells showed higher resistance for membrane permeabilization compared to SCR controls. In line, perforin blockade equalized susceptibility to membrane permeabilization of both, NFAT2 KO cells and controls, confirming the involvement of NFAT2 in maintaining membrane integrity. In addition, caspase 3/7 activation was reduced in NFAT2 KO cells upon exposure to NK cell granule content as well as upon treatment with staurosporine. This shows an important role of NFAT2 for resistance of the leukemic cells at early stages of apoptosis. The results on the role of NFAT2 in resistance to NK effector function were confirmed with primary CLL patient cells, which were grouped according to high and low NFAT2 expression followed by treatment with NK cell content. Our results demonstrate that loss of NFAT2 allows CLLs cells to evade NK cell effector function. This holds true for both, constitutive cytotoxicity and therapeutically induced ADCC and is due to lowered susceptibility of the leukemic cells to perforin mediated membrane permeabilization and intrinsic resistance to granzyme induced apoptosis. Thus, NFAT2 loss, which correlates with aggressive clinical course in patients, facilitates resistance of CLL cells to CD20 antibody-based treatment. Citation Format: Samuel J. Holzmayer, Sarah M. Greiner, Kuebra Kaban, Jonas S. Heitmann, Helmut R. Salih, Melanie Maerklin. NFAT2 determines susceptibility of chronic lymphocytic leukemia cells to constitutive and Rituximab-induced NK cell killing [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 2102.

BCL-2 expression promotes immunosuppression in chronic lymphocytic leukemia by enhancing regulatory T cell differentiation and cytotoxic T cell exhaustion

BackgroundChronic lymphocytic leukemia (CLL) results in increased susceptibility to infections. T cell dysfunction is not associated with CLL in all patients; therefore, it is important to identify CLL patients with T cell defects. The role of B-cell lymphoma-2 (BCL-2) in CLL has been explored; however, few studies have examined its role in T cells in CLL patients. Herein, we have investigated the regulatory role of BCL-2 in T cells in the CLL tumor microenvironment.MethodsThe expression of BCL-2 in T cells was evaluated using flow cytometry. The regulatory roles of BCL-2 were investigated using single-cell RNA sequencing (scRNA-seq) and verified using multi-parameter flow cytometry on CD4 and CD8 T cells. The clinical features of BCL-2 expression in T cells in CLL were also explored.ResultsWe found a significant increase in BCL-2 expression in the T cells of CLL patients (n = 266). Single cell RNA sequencing (scRNA-seq) indicated that BCL-2+CD4+ T cells had the gene signature of increased regulatory T cells (Treg); BCL-2+CD8+ T cells showed the gene signature of exhausted cytotoxic T lymphocytes (CTL); and increased expression of BCL-2 was associated with T cell activation and cellular adhesion. The results from scRNA-seq were verified in peripheral T cells from 70 patients with CLL, wherein BCL-2+CD4+ T cells were enriched with Tregs and had higher expression of interleukin-10 and transforming growth factor-β than BCL-2−CD4+ T cells. BCL-2 expression in CD8+T cells was associated with exhausted cells (PD-1+Tim-3+) and weak expression of granzyme B and perforin. T cell–associated cytokine profiling revealed a negative association between BCL-2+ T cells and T cell activation. Decreased frequencies and recovery functions of BCL-2+T cells were observed in CLL patients in complete remission after treatment with venetoclax.ConclusionBCL-2 expression in the T cells of CLL patients is associated with immunosuppression via promotion of Treg abundance and CTL exhaustion.

TIM-3 in Leukemia; Immune Response and Beyond.

T cell immunoglobulin and mucin domain 3 (TIM-3) expression on malignant cells has been reported in some leukemias. In myelodysplastic syndrome (MDS), increased TIM-3 expression on TH1 cells, regulatory T cells, CD8+ T cells, and hematopoietic stem cells (HSCs), which play a role in the proliferation of blasts and induction of immune escape, has been reported. In AML, several studies have reported overexpression of TIM-3 on leukemia stem cells (LSCs) but not on healthy HSCs. Overexpression of TIM-3 on exhausted CD4+ and CD8+ T cells and leukemic cells in CML, ALL, and CLL patients could be a prognostic risk factor for poor therapeutic response and relapse in patients. Currently, several TIM-3 inhibitors are used in clinical trials for leukemias, and some have shown encouraging response rates for MDS and AML treatment. For AML immunotherapy, blockade TIM-3 may have dual effects: directly inhibiting AML cell proliferation and restoring T cell function. However, blockade of PD-1 and TIM-3 fails to restore the function of exhausted CD8+ T cells in the early clinical stages of CLL, indicating that the effects of TIM-3 blockade may be different in AML and other leukemias. Thus, further studies are required to evaluate the efficacy of TIM-3 inhibitors in different types and stages of leukemia. In this review, we summarize the biological functions of TIM-3 and its contribution as it relates to leukemias. We also discuss the effects of TIM-3 blockade in hematological malignancies and clinical trials of TIM-3 for leukemia therapy.

Immunoglobulin Heavy Chain Variable Region (IgHV) Unmutated, but Not Mutated, Chronic Lymphocytic Leukemia (CLL) Arises in a CD34+ Progenitor

Background: It is now clear that CLL is a heterogeneous disease with varied prognoses. Cytogenetics and IgHV mutational status are 2 of the most important prognostic variables. Recent data from our group and others suggest that IgHV mutational status has a greater impact on prognosis than cytogenetics; accordingly, the small fraction (about 25%) of del(17p) and del(11q) CLL patients with mutated IgHV have relatively favorable prognoses, while those (about 30%) del(13q) patients with unmutated IgHV have relatively unfavorable prognoses. Although CLL with mutated IgHV should arise from a post-germinal center B cell, we studied whether IgHV unmutated CLL could arise as early in B cell differentiation as a CD34+CD19+ cell.Methodology: We prospectively studied 20 patients with overt mutated IgHV CLL and 31 with overt unmutated IgHV CLL for the presence of CD34+CD19+ cells harboring the specific patient's CLL cytogenetic abnormality by fluorescence in situ hybridization (FISH). CD34+ cells were isolated from the patients' peripheral blood by magnetic bead and column (Miltenyi Biotec). The isolated cells were then analyzed by flow cytometry for CD34 and CD19 expression, and CD34+CD19+ cells (if present) were sorted onto slides and FISH performed using probes specific for the patient's known cytogenetic abnormality.Results: Essentially no population of CD34+CD19+ cells could be detected circulating in patients with mutated IgHV (Figure 1, 2 representative patients). However, there was a small (0.32% of circulating cells, range 0.15-1.5%), but easily detectable, population of CD34+ cells in 29/31 patients with unmutated IgHV (Figure 2, 2 representative patients with sorting gates shown) regardless of the cytogenetic abnormality [9 patients with +12, 7 with del(13q), 6 with del(17p), 5 with del(11q), and 4 with other). These CD34+ cells were exclusively CD19+. The 2 unmutated patients without detectable CD34+CD19+ cells both harbored +12 abnormalities. When enough CD34+CD19+ cells were isolated for FISH analysis, the CLL-specific chromosomal abnormality was detected in 99+1% of the CD34+CD19+ cells, including in those patients with del(13q) and unmutated IgHV (Figure 2A).Conclusion: We show that CD34+CD19+ cells harboring the CLL-specific cytogenetic abnormality can be found in the vast majorith of CLL patients with unmutated IgVH, regardless of cytogenetic abnormality. We could not detect these cells in CLL patients with mutated IgHV. These data suggest that the IgHV mutational status may provide a glimpse into at least 2 biologically distinct diseases, both of them currently called CLL. The primitive cell of origin in unmutated IgHV CLL may in part explain its poorer prognosis,and suggests that this form of CLL is indeed a leukemia, arising from the bone marrow. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Functional Implication of p53-Mediated DNA Damage Response on the B-Cell Lymphoma Microenvironment Interaction in Chemoimmunotherapy

Resistance against chemotherapy remains a challenge in the current treatment of B cell malignancies. In previous work we could show that macrophages are an essential element in the synergistic response to the administration of the DNA damaging agent Cyclophosphamide and monoclonal antibodies. The combination treatment strongly increases tumor clearance overall survival in the treatment refractory humanized BCL2-MYC-dependent double hit lymphoma hMB mouse model.Here we observed that the alkylating agent Cyclophosphamide is inducing the secretion of distinct cytokines like VEGF-A, CCL4, IL8, IL10 and TNFα by the leukemic cells. This in turn leads to a repolarization of tumor associated macrophages from a suppressive to an activated phenotypic state, which is designated as acute secretory activating phenotype (ASAP). We analyzed the functional implications of the DNA damage response pathway for the generation of the ASAP and synergy in chemoimmunotherapy. When disrupting DNA damage response pathway by knock-down of key elements like ATM, DNA-PK or p53 in the leukemic cells we could prevent the formation of the stimulatory ASAP effect on macrophage phagocytic capacity. Particularly p53 status seems to possess a central regulating role on macrophage mediated malignant cell depletion. TP53 activation via Nutlin-3A treatment of lymphoma cell enhances ADCP in in p53 wildtype cells, while not displaying enhancement in p53-deficient lymphoma cells. Utilizing the hMB model for modeling in vivo treatment of lymphoma bearing mice we could demonstrate diminished ASAP and ADCP for p53-deficient lymphoma treated with cyclophosphamide in vivo . Using primary human CLL patient cells comparing both wildtype and p53-deficient status, the p53-deficient CLL cells failed to induce the stimulatory, cytokine-mediated effect on macrophage phagocytosis in response to combination treatment as seen with the p53 proficient CLL cells. This observation is underlined by in vivo modeling of CLL therapy by treating Eµ-TCL1/p53wt/wt as well as Eµ-TCL1p53-/- mice. Here, low-dose cyclophosphamide treated Eµ-TCL1p53-/- mice failed to induce an antibody mediated stimulatory effect on macrophage phagocytosis capacity as seen with Eµ-TCL1/p53wt/wt mice.In this work, we are able to recapitulate the effect of ASAP mediated increase of macrophage mediated antibody dependent cellular phagocytosis in the Eµ-Tcl1 mouse model and therefore demonstrate that the ASAP is not only restricted to double hit lymphoma, but also shows functional relevance in low-grade lymphoma such as CLL. Moreover, we indicate that p53 functional status influences the generation of the ASAP in independent models in vitro and in vivo as in primary CLL patient cells. Mediated by this process p53 regulates the interraction with tumor asociated macrophages and the tumor microenvironment.Finally our ongoing research offers possibility to reveal and tailor new treatment approaches for chemorefractory patients. DisclosuresHallek:AbbVie: Consultancy, Honoraria, Research Funding; Amgen: Consultancy, Honoraria, Research Funding; Celgene: Consultancy, Honoraria, Research Funding; F. Hoffmann-LaRoche: Consultancy, Honoraria, Research Funding; Gilead: Consultancy, Honoraria, Research Funding; Janssen-Cilag: Consultancy, Honoraria, Research Funding; Mundipharma: Consultancy, Honoraria, Research Funding.

KIAA0101 knockdown inhibits cell proliferation and induces cell cycle arrest and cell apoptosis in chronic lymphocytic leukemia cells.

BackgroundChronic lymphocytic leukemia (CLL) is a heterogeneous disease with intense cytogenetic aberrations. Importantly, our recent report indicated that thyroid hormone receptor interactor 13 (TRIP13) is a potential new therapeutic target in CLL. In this study, we predicted 20 TRIP13-related genes and found that KIAA0101 is a novel gene that regulates cell proliferation and the cell cycle of CLL cells.MethodsCD19+ B cells were isolated from the peripheral blood of 26 CLL patients and 6 healthy donors through magnetic cell sorting. Cell proliferation was assessed by the CCK-8 assay. The mRNA and protein levels of genes were examined through RT-qPCR and western blot assays, respectively. Cell cycle and cell apoptosis were measured through Annexin V-based flow cytometry and the caspase 3/7 activity assay. Potential targets of KIAA0101 were identified through microarray analysis. 20 TRIP13 related genes was predicted by Ingenuity Pathway Analysis (IPA). KIAA0101-regulated functions and molecular pathways were predicted through IPA.ResultsKIAA0101 knockdown had the strongest inhibitory effect on CLL cell proliferation among the 20 TRIP13-related genes. KIAA0101 was highly expressed in CD19+ B cells of CLL patients. KIAA0101 knockdown induced cell cycle arrest and cell apoptosis, and inhibited FOXO1, MYD88, and TLR4 expression in CLL cells.ConclusionsTaken together, we demonstrated that KIAA0101 plays a critical role in cell proliferation and the cell cycle of human CLL cells. KIAA0101 knockdown induced cell apoptosis, and reduced FOXO1, MYD88, and TLR4 expression, and may therefore be used as a therapeutic target of CLL.

Apoptotic effect of berberine via Bcl-2, ROR1, and mir-21 in patients with B-chronic lymphocytic leukemia.

Berberine is a natural isoquinoline alkaloid that has been shown to inhibit the proliferation and induce apoptosis in a wide variety of tumor cells. However, the action mechanism of berberine in CLL cells is unknown. The previous study has shown that berberine leads to reduced viability and elevated levels of apoptosis in PBMCs of CLL patients. CLL cells are characterized by remarkable expression of Bcl-2 and ROR1 which leads to activation and survival and increases disease progression in patients. High-level expression of miR-21 in patients with CLL is associated with a higher risk of death. Here we investigated the anticancer effects of berberine upon peripheral blood mononuclear cells (PBMCs) of CLL patients. To evaluate the expression of anti-apoptotic proteins and ROR1 using flow cytometry and western blot, PBMCs were treated with 25 μM of berberine for 24 hr. The expression levels of mir-21 were evaluated by real-time PCR. Examination of treated cells demonstrated that berberine decreased Bcl-2 and ROR1 levels. Although western blot results did not show any change in Bax as a pro-apoptotic protein, an increased Bax/Bcl-2 ratio indicated that mitochondrial pathway is involved in berberine-induced apoptosis of CLL cells. Interestingly, berberine could reduce the expression of miR-21 in comparison to the untreated group. Our findings describe some of the molecular mechanisms of berberine by decreasing Bcl-2, ROR1, and mir-21 which may be considered as a novel apoptosis inducer in CLL cells.

DNA Damage-Response Pathway in Lymphoma Determines Interactions with Macrophages By Altered PD-L1 Expression and Exosome Formation

The tumor microenvironment is characterized by multiple interactions of transformed malignant cells with non-transformed stroma or immune cells. Particularly macrophages play a pivotal role in this network determining disease progression and therapeutic response. In previous work we could show that macrophages are an essential mediator of therapeutic response in the synergistic response to the administration of the chemoimmunotherapy. The combination treatment strongly increases tumor clearance by repolarization of tumor-associated macrophages from a suppressive to an activated phenotypic state. Here, se analyzed the functional implications of the DNA damage response pathway for the generation of the ASAP and synergy in chemoimmunotherapy.We attempted to disrupt DNA damage response pathway in lymphoma cells generated from the hMB humanized Double-Hit-Lymphoma model by knock-down of key elements like ATM, DNA-PK or p53. We could prevent the formation of the stimulatory cytokine release effect on macrophage phagocytic capacity. Here, p53 status displays a key regulatory role on macrophage mediated malignant cell depletion. TP53 activation via Nutlin-3A treatment of lymphoma cell enhances ADCP in in p53 wild-type cells, while not displaying enhancement in p53-deficient lymphoma cells. Addressing the treatment in vivo using the hMB model for modeling of Double-Hit Lymphoma bearing mice we could demonstrate diminished ASAP and ADCP for p53-deficient lymphoma treated with cyclophosphamide in vivo.Using primary human CLL patient cells comparing both wild-type and p53-deficient status, the p53-deficient CLL cells failed to induce the stimulatory, cytokine-mediated effect on macrophage phagocytosis in response to combination treatment as seen with the p53 proficient CLL cells. Using a CLL mouse model by treating Eµ-TCL1/p53wt/wt as well as Eµ-TCL1p53-/- mice we could show that low-dose cyclophosphamide treated Eµ-TCL1p53-/- mice failed to induce an antibody mediated stimulatory effect on macrophage phagocytosis capacity as seen with Eµ-TCL1/p53wt/wt mice. A similar effect was seen for primary multiple myeloma cells in response to daratumumab displaying significantly less ADCP of p53-deficient multiple myeloma cells.As for the mechanism of p53-defined interaction within the tumor microenvironment we subjected p53-wild-type and p53-deficient lymphoma cells for proteomic analysis. Here we could identify a significantly deregulated protein expression profile for exosome release in p53 deficient lymphoma cells. Verifying this finding by assessing size and frequency exosomes released by respective cell populations we reveal profound changes induced by p53 loss.Furthermore we could identify up-regulation of PD-L1 in p53-deficient cells. Blocking this checkpoint in the ADCP assay could significantly restore phagocytic capacity of macrophages and overall therapeutic response.In this work, we indicate that p53 functional status determines phagocytic function and therapeutic response to monoclonal antibodies. We can verify this finding in independent models in vitro and in vivo as in primary CLL and myeloma patient cells. We furthermore identify altered exosome profiles and checkpoint inhibitor expression in lymphoma cells as underlying mechanism of macrophage modulation.Finally our ongoing research offers possibility to reveal and tailor new combinatorial treatment approaches for chemo-refractory patients. DisclosuresWendtner:Genetech: Consultancy, Honoraria, Other: travel support, Research Funding; GlaxoSmithKline: Consultancy, Honoraria, Other: travel support, Research Funding; Gilead: Consultancy, Honoraria, Research Funding; Janssen: Consultancy, Honoraria, Other: travel support, Research Funding; Pharmacyclics: Consultancy, Honoraria, Other: travel support, Research Funding; Abbvie: Consultancy, Honoraria, Other: travel support, Research Funding; MorphoSys: Consultancy, Honoraria, Other: travel support, Research Funding; Gilead: Consultancy, Honoraria, Other: travel support, Research Funding; Roche: Consultancy, Honoraria, Other: travel support, Research Funding; Mundipharma: Consultancy, Honoraria, Research Funding. Hallek:Janssen: Honoraria, Research Funding; Celgene: Honoraria, Research Funding; Mundipharma: Honoraria, Research Funding; Pharmacyclics: Honoraria, Research Funding; Roche: Honoraria, Research Funding; Gilead: Honoraria, Research Funding; Abbvie: Honoraria, Research Funding. Pallasch:Gilead: Research Funding.

New insights into smudge cell percentage in chronic lymphocytic Leukemia: A novel prognostic indicator of disease burden

BackgroundPercentage of smudge cells in CLL patients has recently been reported as a novel prognostic factor. ObjectivesTo investigate the impact of smudge cells percentage on the clinicolaboratory data of CLL patients and to evaluate the relationship between it and other prognostic factors in CLL. MethodsNinety adults with CLL were enrolled. Smudge cells percentage was calculated by microscopic evaluation of blood smears. Testing of CD38 expression was done by immunophenotyping and detection of ATM, P53 deletions and trisomy 12 were performed using fluorescent in situ hybridization (FISH) ResultsLower smear cells percentage (<30%) was significantly correlated with age, lymphadenopathy, organomegaly and advanced staging. It was also associated with high TLC, low hemoglobin and platelets count and high absolute and atypical lymphocytic count. Correlation studies with other prognostic factors revealed an association between low smear cells percentage and CD38 expression, short LDT, P53 and ATM deletions. Logistic regression analysis was also done to provide complementary prognostic information identifying the significant independent factors that predict low smear cell percentage. Conclusionlow percentage of smudge cells (<30%) could be considered as an adverse prognostic predictor being associated with high risk markers in CLL.

Ig-like transcript 2 (ILT2) suppresses T cell function in chronic lymphocytic leukemia

ABSTRACTChronic lymphocytic leukemia (CLL) is associated with a profound dysregulation of the immune system. Loss of T cell function is frequently caused in cancer by sustained signaling of inhibitory receptors. Here, we analyzed the role of the novel inhibitory receptor Ig-like transcript 2 (ILT2) in the pathogenesis of CLL. We observed that ILT2 expression was markedly reduced on leukemic cells, whereas it was increased on CD8 and CD4 T cells from CLL patients, particularly in those patients harboring chromosome 11q deletion, which includes the ATM gene. A deep dysregulation of ILT2 ligands expression in leukemia cells was also observed. ILT2 impaired the activation and proliferation of CD4 and CD8 T cells in CLL patients, but it had no effect in leukemic cells. ILT2 downregulated the production of IL-2 by CD4 T cells of CLL patients and induced the expression of cytokines that promote the survival of leukemic cells, such as IFN-γ, by T cells. Importantly, ILT2 blockade restored the activation, proliferation and cytokine production of T cells. In conclusion, we describe a novel immune inhibitory pathway that is upregulated in CLL and delineate a new potential target to be explored in this disease.

A novel inhibitor of BCL2, Disarib abrogates tumor growth while sparing platelets, by activating intrinsic pathway of apoptosis

Antiapoptotic protein BCL2, serves as an excellent target for anticancer therapy owing to its increased level in cancers. Previously, we have described characterization of a novel BCL2 inhibitor, Disarib, which showed selective cytotoxicity in BCL2 ‘high’ cancer cells and CLL patient cells. Here, we have investigated the mechanism of Disarib-induced cytotoxicity, and compared its efficacy with a well-established BCL2 inhibitor, ABT199. We show that Disarib administration caused tumor regression in mouse allograft and xenograft models, exhibited platelet sparing property and did not exhibit significant side effects. Importantly, comparison between Disarib and ABT199, revealed higher efficacy for Disarib in mouse tumor model and cancer cell lines. Disarib induced cell death by activating intrinsic apoptotic pathway. Interestingly, Disarib showed synergism with paclitaxel, suggesting its potential for combination therapy. Thus, we provide mechanistic insights into the cell death pathways induced by Disarib, report that Disarib exhibited better effect than currently used ABT199 and demonstrate its combinatorial potential with paclitaxel.

Abstract A16: Establishment and characterization of CCGL007, a novel human chronic lymphocytic leukemia cell line

Abstract Chronic lymphocytic leukemia (CLL) is a monoclonal disorder characterized by a progressive accumulation of functionally incompetent lymphocytes. However, lack of immortalized cell line representative of the CLL disease has hampered a full understanding of disease pathogenesis and development of novel therapeutic agents for treatment. There were very few representative CLL cell lines are available now. To this end, we have tested different culture condition with addition of growth factor supplements to generate CLL cell lines from highly purified peripheral blood B cells of CLL patients. Recently, we have successfully established a new CLL cell line, named CCGL007. CCGL007 cells grow as suspension in liquid culture. Surface marker analysis with a panel of monoclonal antibodies revealed that CCGL007 cell line expresses B cell markers. The chromosomal, immunophenotypic, molecular biologic characteristics, and in vivo tumorigenesis potential of CCGL007 is under investigation. We have used this cell model to test the efficacy of selected standard chemotherapy drugs and new therapeutic agents, such as ibrutinib and several novel in-house BTK inhibitors, against CLL cell growth, and CCGL007 represents as a suitable preclinical model for testing pharmacological agents. Citation Format: Ching-Chuan Kuo, Chih-Cheng Chen, Chih-Hsiang Huang, Lin Li-Mei. Establishment and characterization of CCGL007, a novel human chronic lymphocytic leukemia cell line. [abstract]. In: Proceedings of the AACR Special Conference: Patient-Derived Cancer Models: Present and Future Applications from Basic Science to the Clinic; Feb 11-14, 2016; New Orleans, LA. Philadelphia (PA): AACR; Clin Cancer Res 2016;22(16_Suppl):Abstract nr A16.

Mono- and dual-targeting triplebodies activate natural killer cells and have anti-tumor activity in vitro and in vivo against chronic lymphocytic leukemia

ABSTRACTChronic lymphocytic leukemia (CLL) is the most common form of leukemia that affects B lymphocytes in adults. Natural killer (NK) cells in CLL patients are intrinsically potent but display poor in situ effector functions. NKG2D is an activating receptor found on NK and CD8+ T cells and plays a role in immunosurveillance of CLL. In this study, we developed mono- and dual-targeting triplebodies utilizing a natural ligand for human NKG2D receptor (ULBP2) to retarget NK cells against tumor cells. Triplebodies in both formats showed better ability to induce NK-cell-dependent killing of target cells compared to bispecific counterparts. A mono-targeting triplebody ULBP2-aCD19-aCD19 successfully triggered NK cell effector functions against CLL cell line MEC1 and primary tumor cells in allogenic and autologous settings. Additionally, a dual-targeting triplebody ULBP2-aCD19-aCD33 specific for two distinct tumor-associated antigens was developed to target antigen loss variants, such as mixed lineage leukemia (MLL). Of note, this triplebody exhibited cytotoxic activity against CD19/CD33 double positive cells and retained its binding features even in the absence of one of the tumor antigens. Further, ULBP2-aCD19-aCD19 showed significant in vivo activity in immune-deficient (NSG) mouse model transplanted with CLL cell line as target cells and human immune cells as an effector population providing a proof-of-principle for this therapeutic concept.

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

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

BCL2-Inhibitors Target a Major Group of Newly-Diagnosed and Relapsed/Refractory Acute Myeloid Leukemia Ex Vivo

BACKGROUND:BCL-2 family members play a critical role in the regulation of apoptosis. BCL-2 and BCL-XL promote cell survival by preventing mitochondrial apoptotic pore formation. BH3 mimetic drugs such as venetoclax (ABT-199) promote apoptosis by inhibiting BCL-2 while navitoclax (ABT-263) inhibits both BCL-2 and BCL-XL. In AML, the expression of anti-apoptotic proteins is highly variable. In a recent study venetoclax showed single-agent activity in 6/12 AML cell lines and 20/25 patient samples. (Pan et al. Cancer Disc 2014). The samples with complex cytogenetics were largely resistant. Sensitivity correlated with increased BCL-2 protein levels and negatively correlated with BCL-XL and MCL-1 protein levels. We aimed to expand these data to both newly diagnosed and relapsed AML patients and to isolate biomarkers for patient selection.METHODS:We assessed the ex vivo sensitivity of fresh leukemic cells from 16 diagnosed and 36relapsed/refractory AML patient samples to venetoclax (25 samples) and navitoclax (52 samples). Exome sequencing was performed on 32 samples and gene expression of BCL2 family members (BCL-2, BCL-XL, MCL-1, BIK, BAX, BAK1, BID, BCL2L12, BIM, BCL2A1, PUMA and BAD) was determined on 31 samples by qRT-PCR. Samples from primary cells of healthy individuals (n=10), and CMML (n=7) or CLL (n=2) patients were used as controls. Drug sensitivity was determined over a 10,000-fold concentration range (1-10 000 nM). A leukemia-specific drug sensitivity score (sDSS) derived from area under the dose response curve calculations was used as the efficacy variable by comparing leukemia results with those from normal bone marrow cells (Bhagwan et al., Sci Rep 2014, Pemovska et al., Cancer Disc 2014).RESULTS:Compared to healthy controls, CMML samples were largely non-sensitive, whereas CLL samples were highly sensitive to BCL-2 inhibitors ex vivo. The AML samples exhibited heterogeneous responses. 15/25 (60%) of AML samples were sensitive to venetoclax and 35/52 (67%) to navitoclax.Both diagnostic (12 of 16 samples, 75%) and relapsed/refractory samples (24 of 36 samples, 64%) were sensitive to navitoclax. Similarly, 6/7 (86%) of diagnostic samples and 9/18 (50%) of relapsed/refractory samples were sensitive to venetoclax. We observed responses to venetoclax and navitoclax in each patient to be similar, although navitoclax showed efficacy at lower concentrations: in 25 samples tested with both agents, mean sDSS values were lower in navitoclax-treated samples (paired t-test, p=0.02). All except one patient sample exhibited a difference in resistance between the two drugs showing sensitivity to navitoclax but not to venetoclax.We observed responses across all mutational profiles, including samples with mutations to FLT3 -ITD, NPM1, TP53, NRAS and IDH1 and IDH2, as well as in samples with complex karyotypes. Intriguingly, three of four samples with mutated TP53 exhibited sensitivity to BCL2 inhibition. No single mutation predicted sensitivity or resistance. At the RNA level, no statistical correlation between BCL2 or BCL-XL expression for BCL2 inhibitor response was observed. Instead we observed high levels of beta-2-microglobulin (B2M) mRNA expression in BCL2 inhibitor-resistant samples with a strong negative correlation to navitoclax sensitivity (r=-0.60, P=0.0008).DISCUSSION:We did not observe BCL2 and BCL-XL mRNA expression to be optimal predictors for BCL2 inhibitor response. On the other hand, we observed high expression of B2M mRNA expression in resistant samples suggesting that it could serve as a biomarker for sensitivity to BCL2 inhibitors. The high B2M expression has been previously linked to poor prognosis in solid tumors and in AML (Albitar et al., Leukemia 2007). In cell line models B2M leads to phosphorylation and inactivation of proapototic protein BAD (Nokura et al., J Urol 2007). This may affect the balance between pro- and antiapoptotic proteins and thus offer a escape route from BCL2 inhibition.To conclude, we observed BCL2 inhibition to be effective ex vivo in over half of all AML samples, tested both in primary and relapsed/refractory state as well as across different subgroups defined by AML driver mutations. Of the potential biomarkers that were assessed, B2M was the best mRNA-level indicator for anti-BCL-2 drug efficacy. DisclosuresOff Label Use: BCL2 inhibitors are not approved for the treatment of AML. Heckman:Celgene: Honoraria, Research Funding; Pfizer: Research Funding. Porkka:Bristol-Myers Squibb: Honoraria; Celgene: Honoraria; Novartis: Honoraria; Pfizer: Honoraria.

A Low Frequency of Losses in 11q Chromosome Is Associated with Better Outcome and Lower Rate of Genomic Mutations in Patients with Chronic Lymphocytic Leukemia.

To analyze the impact of the 11q deleted (11q-) cells in CLL patients on the time to first therapy (TFT) and overall survival (OS), 2,493 patients with CLL were studied. 242 patients (9.7%) had 11q-. Fluorescence in situ hybridization (FISH) studies showed a threshold of 40% of deleted cells to be optimal for showing that clinical differences in terms of TFT and OS within 11q- CLLs. In patients with ≥40% of losses in 11q (11q-H) (74%), the median TFT was 19 months compared with 44 months in CLL patients with <40% del(11q) (11q-L) (P<0.0001). In the multivariate analysis, only the presence of 11q-L, mutated IGHV status, early Binet stage and absence of extended lymphadenopathy were associated with longer TFT. Patients with 11q-H had an OS of 90 months, while in the 11q-L group the OS was not reached (P = 0.008). The absence of splenomegaly (P = 0.02), low LDH (P = 0.018) or β2M (P = 0.006), and the presence of 11q-L (P = 0.003) were associated with a longer OS. In addition, to detect the presence of mutations in the ATM, TP53, NOTCH1, SF3B1, MYD88, FBXW7, XPO1 and BIRC3 genes, a select cohort of CLL patients with losses in 11q was sequenced by next-generation sequencing of amplicons. Eighty % of CLLs with 11q- showed mutations and fewer patients with low frequencies of 11q- had mutations among genes examined (50% vs 94.1%, P = 0.023). In summary, CLL patients with <40% of 11q- had a long TFT and OS that could be associated with the presence of fewer mutated genes.