Lymphocytic Leukemia Cells Research Articles

Abstract 876: Imaging the interaction of leukemia and bone marrow microenvironment in murine model of ALL

Abstract Interactions of leukemia and the bone marrow (BM) microenvironment are known to play a key role in the survival and growth of leukemic cells, and we have shown that HIF-1α stabilization in BM stromal cells facilitates leukemia homing and progression (Chen et al. Blood 2012, 119:4971). Leukemic cells have been shown to hijack the homeostatic mechanisms of normal hematopoietic stem cells (HSCs) and take refuge within the BM niche. This mechanism is pivotal during chemotherapy and contributes to disease relapse. In this study, we aimed to characterize the time-dependent progression of BM hypoxia involving both acute lymphocytic leukemia (ALL) cells and components of the BM niche, using multiphoton intravital microscopy (MP-IVM) . We generated a transplantable, fluorescent leukemia model by retrovirally transducing C57Bl6-Ai14 murine BM cells that express red fluorescing tdTomato with the p190-Bcr/Abl oncogene (KG Harutyunyan et al, Blood 2014 124:2396). The resulting p190-Bcr/Abl tdTomato cells caused rapid development of ALL in non-irradiated C57Bl6 immunocompetent mice, manifested by infiltration of multiple organ and BM sites, followed by death within 14-18 days. We utilized Col2.3-GFPemd transgenic mice as recipients of leukemia to highlight the osteoblastic niche, and visualized vasculature by injection of TRITC-dextran. We showed the dynamic of homing and engraftment of ALL leukemic B cells (LBC) in OB-GFP recipient mice, with homing in the vicinity of blood vessels visualized by MP-IVM, followed by proliferation and leukemia expansion. This was accompanied by invasion of both vascular and osteoblastic components of BM microenvironment. Longitudinal assessment of hypoxia utilizing pimonidazole staining showed progressive development of BM hypoxia starting from Day 10 p.i., paralleling leukemia progression, despite the abundant vascularization of the BM. To assess the integrity of the BM vascular niche during leukemia progression the experimental mice were intravitally injected with low doses of an Alexa-Fluor-647-conjugated VE-cadherin antibody (MG. Poulos et al, Epub 2013 Sep 5) to visualize the number and morphology of the perfused vessels and to analyze the architecture of the hematopoietic compartment. We observed that at late stage of leukemia progression (day 12-14 p.i) BM vessels are disorganized as a result of expansion of leukemic B cells. Ongoing longitudinal imaging experiments will characterize the cellular origin of hypoxic niche cells and the dynamic of vasculature alteration in ALL. In summary, these findings demonstrate rapid development of intra-BM hypoxia that parallels leukemia progression and involves interactions between leukemia cells and BM niche cells, as well as disordered vasculature. Citation Format: Karine G. Harutyunyan, Saradhi Mallampati, Anna Zal, Mateusz Rytelewski, Michael C. Gutkin, Jason M. Butler, Tomasz Zal, Marina Konopleva. Imaging the interaction of leukemia and bone marrow microenvironment in murine model of ALL [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 876. doi:10.1158/1538-7445.AM2017-876

Abstract 5423: ZAP70 alters metabolism in chronic lymphocytic leukemic cells

Abstract Chronic lymphocytic leukemia (CLL) has the highest incidence among adults in western countries. Prognosis of CLL is highly variable but one of the biomarker zeta-chain-associated protein 70 (ZAP70) over expression is associated with aggressive disease with time to treatment being 2.6 years for ZAP70 expressing (ZAP70+) patients and 8 years for ZAP70 deficient (ZAP70-) patients. Thus ZAP70 may play a role in CLL progression. Metabolic reprograming plays a central role in cancer progression and is altered in CLL cells. However, the role of ZAP70 in metabolism in CLL cells is remains unclear. We found that ZAP70 binds to pyruvate kinase M2 (PKM2). PKM2 is key isoform of pyruvate kinase which is rate limiting enzyme of the glycolytic pathway and provides a metabolic advantage to tumour cells allowing for increased growth. Our results also indicated the PKM2 is expressed in CLL cells and binding to ZAP70 is not affected by glucose starvation or treatment with kinase inhibitors. Furthermore immunofluorescence microscopy revealed that ZAP-70 and PKM2 colocalize in CLL cells in the cytoplasm. In addition, we found ZAP70+ cells have increased rate of oxygen consumption from basal level determined by addition of inhibitor (Oligomycin A) and mitochondrial uncoupler (FCCP). Simultaneously ZAP70+ cells also have higher extracellular acidification rates. This indicates ZAP70+ CLL cells have increased mitochondrial respiratory capacity with high glycolytic rate. In addition, fluorometric analysis indicates ZAP70+ CLL cells have high glucose consumption rate towards production of lactate compared to ZAP70- CLL cells. Collectively, our data reveals a new function for ZAP70, which is to regulate the Warburg effect and may play a role in CLL progression and may identify new therapeutic targets. Note: This abstract was not presented at the meeting. Citation Format: Subhadip Das, RF Dielschneider, Elizabeth S. Henson, Spencer B. Gibson. ZAP70 alters metabolism in chronic lymphocytic leukemic cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5423. doi:10.1158/1538-7445.AM2017-5423

Venetoclax for the treatment of patients with chronic lymphocytic leukemia.

Venetoclax is a potent, selective inhibitor of BCL-2, a key regulator of the intrinsic pathway of apoptosis. In preclinical studies, venetoclax bound to BCL-2 with high affinity and rapidly induced apoptosis in chronic lymphocytic leukemia (CLL) cells. In early-phase clinical trials in CLL, venetoclax treatment led to tumor lysis syndrome in some patients with a large tumor burden, but this risk was subsequently mitigated by a revised study design that included lower initial dosing with intrapatient dose ramp up and close tumor lysis syndrome monitoring and prophylaxis. Other toxicities, such as neutropenia and gastrointestinal adverse events, were manageable. Venetoclax monotherapy resulted in durable and deep responses in patients with relapsed, refractory CLL, including for those with deletion 17p, leading to the approval of venetoclax by the US FDA for relapsed or refractory deletion 17p CLL, and recently to additional approvals in Europe and Canada. Trials also suggest that venetoclax induces deeper and more durable responses when used in combination with rituximab, and combination studies with other agents are ongoing. Phase III trials are also underway, and will provide data on the efficacy and safety of venetoclax in combination with monoclonal antibodies and targeted therapies in larger patient populations.

New players in the Bruton’s tyrosine kinase vs. ibrutinib match

The Bruton’s tyrosine kinase, also known as BTK, is a member of the Tec family kinases with a well-characterized role in B-cell receptor (BCR) signaling and B-cell activation. BTK is able to transmit and amplify several signals that are involved in the complex crosstalk between tumor cells and microenvironment. These signals include cytokines and growth factors, chemokine receptors, antigen receptors as BCR and integrins (1). Chronic lymphocytic leukemia (CLL) cells show an increased expression and activation of BTK that with a chain reaction modulates signaling pathways essential for CLL-cells survival, as the protein kinase AKT, extracellular signal-regulated kinase (ERK) and nuclear factor kappa light-chain enhancer of activated B cells (NF-κB) pathways (2). In addition, BTK is involved in chemokine-mediated migration, homing and adhesion of CLL cells (3). Given the known importance of BCR signaling in CLL and the central role of BTK in this pathway, in the recent years a new drug called ibrutinib has been used in clinical practice to induce inhibition of this kinase. Ibrutinib is a relatively selective, irreversible inhibitor of BTK binding covalently the Cys-481 in the ATP binding domain of the kinase. Inhibition of BTK in CLL cells leads to an impressive delocalization of leukemic cells from the protective tissue microenvironment to the periphery interfering with pathogenic mechanism of recirculation and homing. Several studies demonstrate the considerable clinical success of ibrutinib showing a good safety profile determining an improvement of the quality of life of CLL patients (4).

Abstract B21: Novel pateamine A analogs to target pro-survival proteins in chronic lymphocytic leukemia

Abstract Introduction: Dysregulation of protein translation is a common feature of cancer. Because the great majority of oncoproteins turn-over rapidly, thus have an acute need for mRNA translation compared to housekeeping proteins, protein synthesis is a promising avenue to explore for targeted cancer therapy. The marine natural product pateamine A (PatA) inhibits cap-dependent translation initiation by binding to the eukaryotic initiation factor 4A (eIF4A). We have previously synthesized a family of PatA-based small molecules. The lead compound, des-methyl, des-amino pateamine A (DMDAPatA), is more stable and easier to synthesize than the parent natural product, and demonstrated anti-cancer activities in both cell lines and animal models. In this study, we extended the investigations of the activities of DMDAPatA in B cell malignancies, focusing on the primary chronic lymphocytic leukemia (CLL) cells. We propose to target the biological rewiring of the CLL cells, namely the overexpression of short-lived pro-survival proteins that keep these cells from undergoing apoptosis. We hypothesize that a transient inhibition of translation will cause a lethal decrease in those labile pro-survival proteins in CLL cells that will initiate apoptosis. Experimental Procedures: Primary CLL cells were used to screen the PatA analogs for their anti-leukemia activity. Cell viability was analyzed by flow cytometry. Protein translation was measured by tritium labeled leucine incorporation. Protein expression was determined by immunoblotting. Results: Our data showed that DMDAPatA blocked protein synthesis in the CLL cells. This action reduced the short-lived anti-apoptotic proteins Mcl-1 and XIAP, and initiated apoptosis in as short as 4 hr. The levels of the more stable anti-apoptotic proteins Bcl-2 and Bcl-XL were not affected. This is consistent with the selective activity of translation inhibitors toward proteins with rapid turn-over rates. Since DMDAPatA depleted Mcl-1 without affecting Bcl-2, and the Bcl-2 antagonist ABT-199 inhibits Bcl-2 activity but spares Mcl-1, the combination of DMDAPatA and ABT-199 targeted both arms of apoptosis control and killed CLL cells synergistically. Dose reduction index analysis demonstrated mutual potentiation of ABT-199 and DMDAPatA. However, in the presence of human plasma proteins, DMDAPatA lost its potency substantially. A plasma protein binding analysis showed over 99% binding in human plasma, which may hamper its development as a therapeutic agent. To address this issue, we synthesized a new series of PatA analogues with the goal of improving the physical properties and CLL potency. We found that any alteration on the binding domain of PatA, either on eastern half of the macrolide ring or the side chain, resulted in dramatic loss of activity toward the CLL cells. The scaffolding domain on the western side of the ring was more tolerable to modifications which made it feasible to improvement. We proposed to introduce an amino group at either the C2 (alpha-amino variant) or C3 position (beta-amino variant) of the macrolide ring to lower lipophilicity and reduce binding to plasma proteins. This strategy led to the identification of three new leads with potent inhibition of protein synthesis. They exhibited greater cytotoxic potency toward CLL cells than DMDAPatA and lower human plasma protein binding. They also showed better selectivity towards CLL cells over normal lymphocytes compared to the parental compound PatA, demonstrating improved therapeutic index. Conclusions: We have successfully synthesized three new PatA analogs with potent anti-leukemia activities. These compounds hold promise for application to cancers with the right biological context, such as CLL, in which the leukemia cells are addicted to the sustained expression of short-lived oncoproteins for survival. Citation Format: Rong Chen, Mingzhao Zhu, Yuling Chen, Wesley Skillern, Qun Qin, William G. Wierda, Kenneth G. Hull, Daniel Romo, William Plunkett. Novel pateamine A analogs to target pro-survival proteins in chronic lymphocytic leukemia. [abstract]. In: Proceedings of the AACR Special Conference on Translational Control of Cancer: A New Frontier in Cancer Biology and Therapy; 2016 Oct 27-30; San Francisco, CA. Philadelphia (PA): AACR; Cancer Res 2017;77(6 Suppl):Abstract nr B21.

Rola idelalizybu w leczeniu chorych na przewlekłą białaczkę limfocytową

Aktywacja receptora limfocytow B (BCR) w komorkach przewleklej bialaczki limfocytowej (CLL) prowadzi do stymulacji szlakow sygnalowych warunkujących ich przezycie. Kinaza 3-fosfatydylo­inozytolu d (PI3Kd) jest jednym z kluczowych bialek uczestniczących w przekazywaniu sygnalu z BCR i integrującym sygnaly z receptorow chemokinowych, bialka CD40 oraz integryn. Pod­wyzszoną aktywnośc tej kinazy zaobserwowano w szczegolności w nowotworach wywodzących sie z ukladu chlonnego, w tym rowniez CLL. W komorkach CLL PI3Kd reguluje procesy proliferacji, roznicowania, apoptozy, metabolizmu, adhezji i migracji. Idelalizyb (GS-1101, CAL-101) jest obecnie jedynym zarejestrowanym i dostepnym w praktyce klinicznej inhibitorem PI3Kd, ktory wykazal obiecującą skutecznośc w badaniach przedklinicznych i klinicznych. W niniejszej publi­kacji omowiono mechanizmy dzialania, skutecznośc kliniczną oraz profil dzialan niepoządanych leczenia idelalizybem.

Targeting Jurkat T Lymphocyte Leukemia Cells by an Engineered Interferon-Alpha Hybrid Molecule

Background/Aims: Adult T-cell leukemia/lymphoma (ATL) is a very aggressive T cell malignancy that carries a poor prognosis, primarily due to its resistance to chemotherapy and to life-threatening infectious complications. Interferon-alpha (IFNα) has been used in combination with the anti-retroviral drug zidovudine to treat patients with ATL. However, the efficacy of long-term therapy is significantly limited due to the systemic toxicity of IFNα. Methods: We utilized phage display library screening to identify short peptides that specifically bind to Jurkat T lymphocyte leukemia cells. By fusing the Jurkat-binding peptide to the C-terminus of IFNα, we constructed an engineered chimeric IFNα molecule (IFNP) for the treatment of ATL. Results: We found that IFNP exhibited significantly higher activity than wild type IFNα in inhibiting the growth of leukemia cells and inducing cell blockage at the G0/G1 phase. The synthetic IFNP molecule exerted its antitumor activity by upregulating the downstream genes involved in the STAT1 pathway and in apoptosis. Using a cell receptor binding assay, we showed that this Jurkat-binding peptide facilitated the binding affinity of IFNα to the cell surface type I IFN receptor. Conclusion: The isolated Jurkat-binding peptide significantly potentiates the therapeutic activity of IFNα in T lymphocyte leukemia cells. The engineered IFNP molecule may prove to a novel antitumor approach in the treatment of patients with ATL.

Atypical Infections in Chronic Lymphocytic Leukemia and Mantle Cell Lymphoma Patients Treated with Ibrutinib

Ibrutinib is an oral Bruton’s tyrosine kinase inhibitor with clinical efficacy in several B cell malignancies. In a pooled analysis of clinical trials evaluating ibrutinib, grade 3-4 infections were reported in 14% of patients. We screened consecutive patients on ibrutinib therapy for chronic lymphocytic leukemia (CLL) or mantle cell lymphoma (MCL) for infectious complications. Patients were hospitalized with documented infections uncommon to this patient population including Legionella pneumonia, Campylobacter bacteremia and fatal progressive multifocal leukoencephalopathy (PML). All patients received prior therapies for CLL/MCL. Immunological evaluation of these patients demonstrated immunoglobulin depression and profound lymphopenia with severe depletion in both B and CD4 T cell counts, suggesting defects in both humoral and T cell mediated immunity. This data support a broader immunosuppressive effect for ibrutinib than initially anticipated. While responding patients may show later recovery of B cell function, profound immunosuppression may occur at earlier treatment time points, particularly in previously treated patients. Closer patient monitoring during the first months of ibrutinib therapy may be desirable.

Unexpected effects of biphosphonates in in vitro models of activated CLL cells

Recent studies suggest that the commonly prescribed anti-osteoporosis drugs bisphosphonates (BPs) might also exhibit antitumor activity. We investigated a possible anticancer effect of BPs on B-chronic lymphocytic leukemia (CLL) cells obtained from peripheral blood of 26 CLL patients. Zoledronate, etidronate and clodronate were administered in vitro simultaneously to following activation stimuli: i) CD40L-expressing fibroblasts, ii) soluble recombinant CD40L produced in our laboratory +IL-4, iii) CpG ODN 2006+IL-15 with or without bone marrow stromal cells (BMSC). CLL cell viability, activation/proliferation were monitored by flow cytometry. We unexpectedly observed that BPs generated a protective effect from spontaneous apoptosis in 11/26 (42%) patients (viability + 18%-392%) and an augmentation in CLL cell activation/proliferation in 61% of the samples (S+G2M phase: +100%±25). Interestingly, protection from spontaneous apoptosis or increment of cell activation, required the presence of either fibroblasts, BMSC or autologous Nurse Like Cells (NLC). We thus hypothesized that supportive cells are involved in the BPs effects either through cell-cell interactions with leukemic cells or T cells, or through soluble factors release in the medium. Functional experiments with transwells suggest that stromal cells, in presence of Clodronate, release soluble factors in the medium that may probably concur to the unexpected Clodronate-mediated enhancement of CLL cell activation/proliferation. This work is in progress and several critical questions on the mechanisms are still unanswered. Nevertheless, the phenomenological data argue that caution should be taken when administering BPs against osteoporosis in elderly persons, who could have Monoclonal B Lymphocytosis or CLL.

Characteristics of splenic marginal zone lymphoma: Clinical, hematologic, and flow cytometry findings of 34 cases

Background: Splenic marginal zone lymphoma (SMZL) is a low-grade disorder that regularly presents with peripheral blood (PB) involvement. A precise description of clinical, laboratory features and immunophenotypic characterization of SMZL are still lacking. Here, we reviewed 34 patients presenting with SMZL to describe the clinical, hematologic features, and flow cytometry immunophenotypic findings of this type of lymphoma at diagnosis. Objectives: The aim of this study is to confirm that SMZL has a specific immunologic profile which enables the hematopathologist and clinician to differentiate this low-grade B-cell lymphoma from other B-cell lymphoproliferative disorder, especially chronic lymphocytic leukemia and hairy cell leukemia, which could sometimes stimulate SMZL morphologically and to emphasize that a correlation of immunophenotypic findings, clinical, and hematologic features of patients plus careful morphological examination of PB and/or bone marrow (BM) aspirate can lead confidently to the correct diagnosis. Materials And Methods: Flow cytometry immunophenotypic findings of 34 cases of SMZL were reviewed. The analysis was performed by BD FACS Calibur™ and FACSCanto II flow cytometers. B lymphocytes were identified according to their Side-Scattered (SSC)/CD19 distribution. A marker was considered positive when expressed in more than 20% of cells above the control. Results: Median age was 60 years, range (35–84 years), both sexes were affected equally. All patients presented with splenomegaly, 71% of patients had absolute lymphocytosis and 88% of patients showed PB involvement. Seventy-four percent of patients had anemia and (53%) of them had thrombocytopenia. Cells from all cases expressed pan B-cell antigens (CD19, CD20), 74% of cases expressed CD79b and Human Leukocyte Antigen – antigen D Related (HLA-DR) expressed in nearly almost all cases (97%). Half of the patients expressed CD11c and SIgD, 41% expressed CD5 and FMC7 while CD25 and CD103 showed positivity in less than 5% of cases. Preferential expression of Kappa light chain was demonstrated, CD10 and CD38, SIgG were negative. Conclusion: SMZL has a distinct immunologic profile which if correlated with morphologic findings of PB or BM aspirates, clinical and hematologic features can help to make the accurate diagnosis and lessens the need of further invasive diagnostic procedure.

Monocytic Leukemia Associated Antigen-34 Mediates Extramedullary Infiltrationand and Tumor Invasion in Acute Myeloid Leukemia

Background: Acute monocytic leukemia(AML-M5), a common hematological malignancy, is serious harm to human health with dangerous clinical prognosis and extramedullary infiltration. Monocytic leukemia associated antigen-34(MLAA-34) gene (GenBank no. AT288977.2) was first discovered in M5 in an effort to identify monocytic leukemia associated antigens by serological identification of antigens by recombinant expression cloning (SEREX). Studies showed that MLAA-34 gene was a new anti-apoptotic molecule associated with acute monocytic leukemia cell U937 through JAK/STAT3 and Wnt signal pathways. Previous clinical studies showed that MLAA-34 was highly expressed in M5 patients involved with extramedullary infiltration and poor survival rate. Further researches on MLAA-34 revealed that MLAA-34 protein expressed in very few cases of lung, colon and stomach cancer and overall survival of these patients were significantly poorer.Objective: This study aims to explore the expression of MLAA-34 in variety of acute myeloid leukemic patients and its correlation with extramedullary infiltration so as to further identify mechanisms of tumor invasive of MLAA-34.Method: Collected bone marrow and peripheral blood samples of 52 newly diagnosed acute myeloid leukemic patients and 14 healthy volunteers, then RT-PCR based on Taqman probe technology was constructed to detect MLAA-34 mRNA and found the relationship between the MLAA-34 expression and extrameduulary infiltration. Blood cell lines including monocytic leukemia cells(THP1, U937), promyelocytic leukemia cell(NB4), myeloid leukemia cell(K562), lymphocytic leukemia cells(RS4;11, Jurkat), myeloma cells(RPMI8226, U266,H929)and solid tumor cells including hepatoma carcinoma cells(MHCC97H, Hep3B, SMMC7721), colon cancer cells(SW480, SW620), breast cancer cells(MCF7, MDA-MB 231), lung carcinoma cells(A549, PC9, H1975) were tested. The total RNA was extracted from cells and patients by TRIzol Reagent (Invitrogen). RT-PCR and Western blot were performed to detect MLAA-34 mRNA and protein express. Short hairpin RNAs (shRNA) targeting MLAA-34 gene were designed and successfully transfected into U937 cells. The efficient knockdown MLAA-34 in U937 cells were constructed to observe MLAA-34 effect on tumor metastasis by transwell invasion experiment.Results: We focused on MLAA-34 gene expression in different subtypes of acute myeloid leukemia (AML). M5 and acute myelomonocytic leukemia (M4) patients expressed high levels of MLAA-34 mRNA, which accompanied with high rates of extramedully lesion. The group of extramedullary infiltration patients presented significantly higher levels of MLAA-34 mRNA than those who did not(P<0.05). MLAA-34 mRNA and protein detected in monocytic leukemia cells(U937 and THP1) showed much higher expression levels than other blood cells and solid tumor cells. The expression of MLAA-34 were generally poor in solid tumor cells, but the high invasive solid tumor cells including colon cancer cell(SW620), hepatocarcinoma cell(MHCC97-H), breast cancer cell(MDA-MB 231) had significant higher levels than equivalent cells with low invasive ability(Figure 1). Transwell experiments detecting the invasion differences among acute myeloid leukemia cells including U937, THP1, NB4 and K562 showed that the level of MLAA-34 mRNA were positively correlated with the cell invasive ability(Figure 2). The expression vectors of MLAA-34 shRNA were successfully constructed. The counts of shRNA-MLAA-34 U937 cells significantly reduced in the lower chambers under microscope(Figure 3). These data revealed that cell invasion ability was significantly correlation with MLAA-34 level and MLAA-34 might mediate tumor invasion.Conclusion: MLAA-34 plays an important role in extremedullay infiltration and MLAA-34 is specifically positively correlated with the invasive ability of cancer cell. [Display omitted] [Display omitted] [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Abstract B041: Ibrutinib treatment counteracts the immunosuppressive activity of malignant B cells

Abstract 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&amp;lt; 0.001) in the frequency of cells producing IL-10 by cycle 3, and a fivefold reduction (P&amp;lt; 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&amp;lt;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&amp;lt;0.001 and P&amp;lt;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.

Abstract 268: PARP3 inhibitors in cancer therapy

Abstract Poly-ADP-ribose-polymerase (PARP1) is an important regulator of DNA damage response (DDR). After induction of certain types of DNA damage, including nicks and double strand breaks (DSB)s, PARP1 is rapidly recruited to altered DNA sites were its catalytic activity produces protein-conjugated long-branched-poly-ADP-ribose (PAR) chains. These protein modifications result in the recruitment and activation of multiple proteins involved in DNA repair. Although there is considerable research describing the characteristics and activity of PARP1/2, there are 15 other PARP family members that are less well characterized. Of these PARP3 has been shown to have a distinct role in DNA repair. This protein has been shown to have a critical role in DSB resolution and to interact with partner proteins know to function in classical nonhomologous end joining (NHEJ) such as DNA-PKcs, Ku70, and Ku80. In order to examine the role of PARP3 vis-à-vis chemotherapy, we utilized the specific PARP3 inhibitor, ME-0328 in combination with activated cyclophosphamide (4HC) in the chronic lymphocytic leukaemia (CLL) cell lines, MEC1 and MEC2. Results from MTT cytotoxicity assays showed that the 50% inhibitory concentration (IC50) (50% of control) of 4HC in MEC-1 cells was 12.9μM. When used in combination with a nontoxic concentration (2μM) of the specific PARP3 inhibitor ME-0328 the IC50 of 4HC was reduced to 3.3μM. In MEC-2 cells the IC50 of 4HC was 10.8μM and was reduced to 2.1μM in the presence of ME-0328 at 2μM. ABT-888, a specific PARP1/2 inhibitor in clinical trials had a minimal effect on 4HC cytotoxicity in these cell lines. 4HC results in interstrand crosslinks (ICLs) which are believed to be repaired by homologous recombination. Intriguingly, PARP3 negatively regulates class switch recombination via activation-induced cytidine deaminase in B-lymphocytes. This may be involved in ME-0328 sensitization of 4HC in B-lymphocytic malignancies. Given these preliminary results we hypothesize that the PARP3 inhibitor, ME-0328 will sensitize B-lymphocytic cancers to chemotherapies. To explore this hypothesis this project will pursue the following objectives: 1. Characterize the selective sensitization of ME-0328 with 4HC and/or bendamustine (an ICL-inducing drug utilized in the treatment of B-lymphocyte malignancies) in B-lymphoma and CLL cell lines plus CLL clinical samples. 2. Determine the mechanisms by which ME-0328 sensitizes CLL cells to 4HC and/or bendamustine (i.e. reduction of PARP1 or 3 activity and/or altered activity of known DNA repair proteins (Rad51, DNA-PK, H2AX) and/or altered activation-induced cytidine deaminase). 3. Determine the in-vivo activity of 4HC and/or bendamustine with or without ME-0328 in a Rag2 /-γcc-/- xenograft model using the MEC1-CLL cell line. The inhibition of PARP3 may increase the sensitivity of tumor cells to DNA damaging chemotherapies. Our results should stimulate development of specific PARP3 inhibitors for clinical use. Citation Format: Bahram Sharif-Askari, David Davidson, Lilian Amrein, Lawrence Panasci. PARP3 inhibitors in cancer therapy. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 268.

Abstract 3554: Functional disparity among BCL-2 members in tonsillar and leukemic B cell subsets probed by next generation BH3 mimetic profiling

Abstract Introduction and aim. Long-lived humoral immunity, in the form of plasma cells (PC) and memory B cells, as well as most B cell malignancies, originate in the germinal center (GC). For successful therapies against malignant B cells it is crucial to understand their survival requirements in relation to their normal counterparts. It was previously shown that murine GC B cells depend on pro-survival protein MCL-1, but not BCL-XL, although both are expressed. In contrast, naïve and memory B cells express BCL-2, and as a consequence, they are sensitive to the BH3 mimetic ABT-737, which blocks both BCL-2 and BCL-XL but not MCL-1. The divergent roles of MCL-1 and BCL-XL in GC B cells still remain unexplained, and it is unknown if this also holds for human B cells. We here dissect these aspects in human tonsillar B subsets and in chronic lymphocytic leukemia (CLL) cells, a B cell cancer where expression of BCL-2 members is influenced by signals that also occur in the normal GC. Experimental procedures. We used FACS sorting of tonsil B subsets, mRNA and protein profiling, co-culture of CLL cells, co-IP of BCL-2 members, cell death assays with next generation BH3 mimetics specific for BCL-2, BCL-XL or MCL-1 (ABT-199, WEHI-539, A-1210477). Results. Naive and memory B cells are mainly sensitive to specific inhibition of BCL-2 by ABT-199. In contrast, GC B cells and tonsil PC are insensitive to ABT-199, but undergo apoptosis when MCL-1 is inhibited. Tonsil PC display yet another profile and are remarkably sensitive to inhibition of BCL-XL. MCL-1 expression in GC B cells is regulated post-translationally and its physiological importance is highlighted by exclusive binding to pro-apoptotic BIM. In contrast, BCL-XL is transcriptionally induced in the GC-light zone, and is solely bound to the weak BH3-only sensitizer BIK, which may explain why BCL-XL is not required for GC B cell survival. These approaches were extended to primary CLL cells either resting or stimulated via CD40, as model for circulating and lymph node resident cells. We showed recently that CD40 stimulation strongly induces MCL-1, BCL-XL and BFL-1, similar to the pattern in healthy GC B cells, and confers complete resistance to ABT-199, whereas untreated CLL cells are highly sensitive. We now demonstrate that dual or triple BH3 mimetic combinations are effective in killing CD40-stimulated CLL. In contrast to GC B cells, BIK is absent in CLL cells while NOXA is highly expressed and, like BIM, is bound to MCL-1. This may account for differential sensitivity to (combinations of) BH3 mimetics. Conclusion. Using novel BH3 mimetics and interaction profiles, we were able to probe the contribution of individual BCL-2 members in survival of normal and malignant B cells. Our findings provide clues to determine therapeutic windows in novel treatment strategies in B cell and other malignancies. Citation Format: Victor Peperzak, Rachel Thijssen, Hanneke ter Burg, Erik Slinger, Arnon P. Kater, Eric Eldering. Functional disparity among BCL-2 members in tonsillar and leukemic B cell subsets probed by next generation BH3 mimetic profiling. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3554.

Abstract 886: Notch signaling activates B-cell specific enhancers to drive oncogene targets in B-cell lymphoma

Abstract Recurrent gain-of-function mutations in genes encoding Notch receptors are associated with poor clinical outcome in chronic lymphocytic leukemia (CLL) and mantle cell lymphoma (MCL), but genome-wide functional targets of Notch signaling in B cells have not been characterized. We identified genome-wide binding sites for Notch transcription factor (TF) complexes, using antibodies specific for intracellular NOTCH1 and its binding partner RBPJ, in CLL and MCL lymph node biopsies, and lymphoma cell lines. We employed a gamma-secretase inhibitor washout strategy to rapidly modulate mutant NOTCH1 protein activation in complementary lymphoma cell line models of ligand-dependent and ligand-independent Notch signaling, and performed RNA-sequencing and genome-wide enhancer acetylation profiling in both the “notch on” and “notch off” states. These data identify a common set of functional Notch target genes in B-cell lymphoma that includes both canonical Notch targets as well as B-cell-specific target genes. The latter are frequently associated with adjacent B-cell-specific enhancers that show direct Notch TF binding and Notch-signaling-dependent histone acetylation. Notch-activated target genes include MYC and other TFs implicated in B-cell lymphoma, and are significantly enriched for mediators of targetable oncogenic lymphoma signaling pathways, (B-cell receptor, toll-like receptor, JAK-STAT, MAP kinase, and G-protein signaling pathways, all FDR q-value &amp;lt; 1e-3 in KEGG and / or Reactome analyses). Many B-cell Notch target genes show significantly increased expression in lymph nodes from patients with NOTCH1 mutant vs. wild-type CLL. Notch target genes are also up-regulated in lymph node- versus peripheral blood-derived CLL cells, consistent with immunohistochemical evidence for microenvironment-dependent Notch signaling activation in the CLL lymph node. Combined small-molecule inhibition of Notch and B-cell receptor signaling showed a synergistic anti-proliferative effect in Notch-dependent lymphoma cells. Our data reveal the functional regulome of Notch signaling in small B-cell lymphoma, and may have implications for the role of Notch signaling in normal B-cell development. These findings suggest novel strategies for rational combination therapy in CLL and MCL. Citation Format: Russell J. Ryan, Jelena Petrovic, Dylan M. Rausch, Winston Lee, Michael Kluk, Laura Donohue, Shawn Gillespie, Jon C. Aster, Warren S. Pear, Bradley E. Bernstein. Notch signaling activates B-cell specific enhancers to drive oncogene targets in B-cell lymphoma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 886.

CD126 and Targeted Therapy with Tocilizumab in Chronic Lymphocytic Leukemia.

IL6 promotes tumor growth and signal transduction via both its membrane-bound (CD126) and soluble receptors (sCD126). We aimed to study whether the levels of CD126 expression in chronic lymphocytic leukemic (CLL) cells can predict in vitro and in vivo treatment response. The levels of membrane-bound CD126 expression were determined on freshly isolated CLL B cells (n = 58) using flow cytometry. These CLL cells were treated with chlorambucil or fludarabine with or without anti-CD126 antibody tocilizumab for 24 hours and IL6-mediated STAT3 transcriptional activity and cell-cycle alteration were evaluated. CD126 surface expression was found in all cases and positively correlated with the levels of in vivo constitutive STAT3 activity. The levels of CD126 expression were significantly and positively correlated with the resistance of CLL cells to in vitro treatment with chlorambucil or fludarabine and poor in vivo treatment response of CLL patients. Blocking IL6 signaling with the anti-CD126 antibody, tocilizumab, had profound effects on STAT3-mediated survival and growth signals: decreased Mcl-1 and Bcl-xL, favoring an apoptotic profile; and decreased p27 with increased cyclin E and CDK2 expression, leading to cell-cycle shift from G0-G1 These tocilizumab-mediated changes induced chemosensitization in resistant CLL cells, with the greatest effect seen in cells with higher CD126 expression (P < 0.001). CLL cells with higher CD126 expression are more resistant to treatment in vivo and in vitro via IL6-CD126-STAT3 axis. Blocking CD126 using tocilizumab sensitizes CLL cells to chemotherapy. Clin Cancer Res; 22(10); 2462-9. ©2015 AACR.

Epigenetic silencing of miR-26A1 in chronic lymphocytic leukemia and mantle cell lymphoma: Impact on EZH2 expression

ABSTRACTDownregulation of miR26A1 has been reported in various B-cell malignancies; however, the mechanism behind its deregulation remains largely unknown. We investigated miR26A1 methylation and expression levels in a well-characterized series of chronic lymphocytic leukemia (CLL) and mantle cell lymphoma (MCL). From 450K methylation arrays, we first observed miR26A1 (cg26054057) as uniformly hypermethylated in MCL (n = 24) (all >75%), while CLL (n = 18) showed differential methylation between prognostic subgroups. Extended analysis using pyrosequencing confirmed our findings and real-time quantitative PCR verified low miR26A1 expression in both CLL (n = 70) and MCL (n = 38) compared to normal B-cells. Notably, the level of miR26A1 methylation predicted outcome in CLL, with higher levels seen in poor-prognostic, IGHV-unmutated CLL. Since EZH2 was recently reported as a target for miR26A1, we analyzed the expression levels of both miR26A1 and EZH2 in primary CLL samples and observed an inverse correlation. By overexpression of miR26A1 in CLL and MCL cell lines, reduced EZH2 protein levels were observed using both Western blot and flow cytometry. In contrast, methyl-inhibitor treatment led to upregulated miR26A1 expression with a parallel decrease of EZH2 expression. Finally, increased levels of apoptosis were observed in miR26A1-overexpressing cell lines, further underscoring the functional relevance of miR26A1. In summary, we propose that epigenetic silencing of miR26A1 is required for the maintenance of increased levels of EZH2, which in turn translate into a worse outcome, as shown in CLL, highlighting miR26A1 as a tumor suppressor miRNA.

A novel class of piperidones exhibit potent, selective and pro-apoptotic anti-leukemia properties.

In the present pre-clinical study, a series of 1-[3-(2-methoxyethylthio)-propionyl]-3,5- bis(benzylidene)-4 piperidones and structurally-related compounds were observed to be cytotoxic in vitro to three human leukemia cell lines, namely Nalm-6, CEM and Jurkat. The 50% cytotoxic concentration (CC50) values of the three cell lines ranged between 0.9-126.4 µM and 0.3-11.7 µM at 24 and 48 h subsequent to exposure, respectively. The two lead compounds with sub-micromolar CC50 concentrations, 1-(2-methoxyethylthio-propionyl)-3,5-bis(benzylidene)-4 piperidone (2a) and 3,5-bis(4-fluorobenzylidene)-1-[3-(2-methoxyethyl sulfinyl)-propionyl]-4-piperidone (3e), were selected for additional analyses. Several strategies were undertaken to determine whether the above piperidones caused cell death via apoptosis or necrosis on T-lymphocyte leukemia Jurkat cells. The results revealed that the two piperidones caused phosphatidylserine externalization, mitochondrial depolarization and activation of caspase-3, which are all biochemical hallmarks of apoptosis. In addition, the selected piperidones displayed selective cytotoxicity towards leukemia cells, and were less toxic in non-cancerous control cells. Therefore, the findings of the present study revealed that the novel piperidones 2a and 3e exert a selective cytotoxic effect on lymphocyte leukemia cells by favoring the activation of the intrinsic/mitochondrial apoptotic pathway.

Serum-soluble APRIL influences survival in patients with chronic lymphocytic leukemia

Background A proliferation-inducing ligand (APRIL) is a member of the tumor necrosis factor family. APRIL plays an important role in B-cell development; it activates chronic lymphocytic leukemia (CLL) cells by reacting with its receptors, enhancing immune recognition, proliferation, and survival of the leukemia cells. Aim The aim of this study was to determine the impact of serum-soluble APRIL levels detected at diagnosis in B-CLL patients, and their relationship with disease parameters and patient outcomes. Patients and methods Serum APRIL levels were estimated in 40 untreated B-CLL patients and 40 apparently healthy controls. Serum was withdrawn at diagnosis of untreated CLL disease and was tested by enzyme-linked immunosorbent assay. Results APRIL levels in peripheral blood samples were significantly higher in B-CLL patients than in normal participants. There were positive correlations between serum-soluble APRIL level, age, white blood cell count, peripheral blood lymphocyte count, CD38%, ZAP-70, and modified RAI staging. There were negative correlations between serum-soluble APRIL, hemoglobin level, and platelet count. During a 2-year follow-up, APRIL levels correlated with overall survival and disease-free survival. Conclusion APRIL is a powerful prognostic marker related to parameters of disease activity and staging and, more importantly, to overall survival and disease-free survival.

Relevance of fatty acid metabolism in proliferating CLL cells

Chronic lymphocytic leukemia (CLL) cells undergo, during their life, iterative cycles of re-activation and subsequent clonal expansion. We previously demonstrated that the antidiabetic drug metformin, known to also inhibit oxidative phosphorylation (OXPHOS), inhibits cell cycle entry of leukemic cells derived ex-vivo from the peripheral blood of CLL patients and stimulated in vitro by cell culture systems that recreate a microenvironment to drive their proliferation (Bruno et al, Oncotarget, 2015). However, overtly proliferating CLL cells were resistant to the cytostatic effects of metformin. Since metformin switched the energetic metabolism of activated, not yet proliferating, CLL cells from OXPHOS to accelerated glycolysis, in the present study we asked whether combining metformin with glycolysis impairment could inhibit also proliferating CLL cells. Still, CLL cells recovered from a transitory block and rescued in vitro proliferation. What kind of energetic reprogramming was involved in the resistance of proliferating CLL cells to glucose utilization? Recent studies highlight on the role of fatty acid utilization of CLL cells. We asked 1) whether inhibitors of lipid metabolism could impair proliferation of in vitro stimulated CLL cells; 2) whether impairing glucose energetic pathways could act synergistically with beta oxidation inhibitors.We found that inhibitors of critical steps of fatty acid metabolisms, such as carnitine-palmitoyl transferase 1A (CPT1A) -rate-limiting enzyme for fatty acid import into mitochondria- or Peroxisome Proliferator-Activated Receptor (PPAR)-alpha -regulator of beta-oxidation- administered at clinically achievable doses, were ineffective on quiescent CLL cells and on CLL cells stimulated by the microenvironment during the first stages of activation. Conversely, remarkable susceptibility to undergo apoptosis was observed at later stages of cell activation and during overt proliferation. Synergism with impairment of other energetic pathways occurred depending on the stage of activation of the in vitro stimulated CLL cells.The results suggest that energetic metabolic pathways could be relevant targets for CLL treatment, provided that the complex metabolic reprogramming network during the transition of leukemic cells from quiescence to proliferation, and back, are clearly elucidated. This work was supported by grants from AIRC IG15426.