Elimination Of Malignant Cells Research Articles

The relationship between immune-related adverse events during ipilimumab monotherapy and survival outcomes among melanoma patients: A systematic review.

Ipilimumab disinhibits immune system activity which results in the elimination of malignant cells. An unintended consequence of ipilimumab therapy is off-target immune-related adverse events (irAEs). It has therefore been proposed that the incidence of irAEs is a manifestation of treatment effectiveness. The objective of this systematic review is to examine the relationship between irAEs and survivability among melanoma patients administered ipilimumab monotherapy. A comprehensive search was conducted across several databases which yielded a total of 2381 studies. Clinical trials and prospective studies administering ipilimumab monotherapy to melanoma patients were included. Furthermore, there was no restriction placed on publication date. After screening, five studies were included for data extraction. The primary outcome of median overall survival (OS) and the secondary outcome of OS hazard ratio were extracted from the included studies. Based on qualitative analysis of the included studies, there seemed to be an association between the occurrence of non-lethal irAEs and improved survival outcomes among melanoma patients administered ipilimumab monotherapy. With that being said, the poorer survivability among patients who experienced high-grade irAEs may be the result of subsequent treatment discontinuation. Potential confounders such as corticosteroid use should be accounted for. Finally, landmark analyses may be conducted to account for immortal time bias. The findings from this systematic review provide evidence suggesting that the incidence of irAEs is a marker of an improved anti-tumor response.

Cord blood derived NK cells activated in counter with tumor cells.

NK cells are initially known for their ability to kill tumor cells with no prior sensitization. Production of mature and long lasting NK cells from Umbilical Cord Blood (UCB) by using cytokines could be a promising method for immunotherapy. NK cells were generated from cord blood cells using IL2, IL7, and IL15 cytokines and measured expression of CD57 and NKp46 markers. Afterward, their capacity in the elimination of malignant cells (Reh cell line) was evaluated by assessment of interferon-γ (as cytokine production sign) and CD107-a expression (as cytotoxic function symptom) using flow cytometry. Our results showed efficient NKp46 + , and CD57 + NK cells generated on day 14. Also, expression of CD107-a and IFN-γ following co-culture with Reh cell lines significantly increased in comparison to the control. Taken together, we have reported one of the best culture conditions for the generation of CD57 + NK cells with on feeder cells and showed appropriate capacity in counter reh cell lines as a target.

Compromised activity of natural killer cells in diffuse large b-cell lymphoma is related to lymphoma-induced modification of their surface receptor expression.

While natural killer (NK) cells are essential players in detection and elimination of malignant cells, these surveillance properties can be compromised by cancer cells. Since NK cell education primarily occurs in the bone marrow and lymphoid tissue, this process might be particularly affected by their infiltration with lymphoma cells. This study aimed to explore functional properties of diffuse large B-cell lymphoma (DLBCL) patient NK cells, which could potentially promote tumour immune evasion and disease propagation.NK cells isolated from the peripheral blood (PB) of 26 DLBCL patients and 13 age-matched healthy controls (HC) were analysed. The cytotoxic CD56dim subtype was the only one identified in patients. Compared to HC, patient cells demonstrated low levels of inhibitory CD158a/b along with decreased expression of activating NKG2D and CD161 and increased inhibitory NKG2A levels. Patient NK cell cytotoxic activity was impaired, as were their degranulation and inflammatory cytokine production, which partially recovered following non-receptor-dependant stimulation.The phenotypically skewed and restricted population of patient NK cells, along with their blunted cytotoxic and immune-regulatory activity, appear to be driven by exposure to lymphoma environment. These NK cell functional aberrations could support lymphoma immune evasion and should be considered in the era of cellular therapy.

Differential role of HLA-A and HLA-B, C expression levels as prognostic markers in colon and rectal cancer

PurposeThe association of human leucocyte antigen (HLA) class I expression levels with the clinical course of many malignancies reflects their crucial role in the recognition and elimination of malignant cells...

Demethylating therapy increases anti-CD123 CAR T cell cytotoxicity against acute myeloid leukemia

Successful treatment of acute myeloid leukemia (AML) with chimeric antigen receptor (CAR) T cells is hampered by toxicity on normal hematopoietic progenitor cells and low CAR T cell persistence. Here, we develop third-generation anti-CD123 CAR T cells with a humanized CSL362-based ScFv and a CD28-OX40-CD3ζ intracellular signaling domain. This CAR demonstrates anti-AML activity without affecting the healthy hematopoietic system, or causing epithelial tissue damage in a xenograft model. CD123 expression on leukemia cells increases upon 5′-Azacitidine (AZA) treatment. AZA treatment of leukemia-bearing mice causes an increase in CTLA-4negative anti-CD123 CAR T cell numbers following infusion. Functionally, the CTLA-4negative anti-CD123 CAR T cells exhibit superior cytotoxicity against AML cells, accompanied by higher TNFα production and enhanced downstream phosphorylation of key T cell activation molecules. Our findings indicate that AZA increases the immunogenicity of AML cells, enhancing recognition and elimination of malignant cells by highly efficient CTLA-4negative anti-CD123 CAR T cells.

In Vitro Interaction of 5-Aminoorotic Acid and Its Gallium(III) Complex with Superoxide Radical, Generated by Two Model Systems.

Increased levels of the superoxide radical are associated with oxidative damage to healthy tissues and with elimination of malignant cells in a living body. It is desirable that a chemotherapeutic combines pro-oxidant behavior around and inside tumors with antioxidant action near healthy cells. A complex consisting of a pro-oxidant cation and antioxidant ligands could be a potential anticancer agent. Ga(III) salts are known anticancer substances, and 5-aminoorotic acid (HAOA) is a ligand with antioxidant properties. The in vitro effects of HAOA and its complex with Ga(III) (gallium(III) 5-aminoorotate (GaAOA)) on the in vitro accumulation of superoxide and other free radicals were estimated. Model systems such as potassium superoxide (KO2), xanthine/xanthine oxidase (X/XO), and rat blood serum were utilized. Data suggested better antioxidant effect of GaAOA compared to HAOA. Evidently, all three ligands of GaAOA participated in the scavenging of superoxide. The effects in rat blood serum were more nuanced, considering the chemical and biochemical complexity of this model system. It was observed that the free-radical-scavenging action of both compounds investigated may be manifested via both hydrogen donation and electron transfer pathways. It was proposed that the radical-scavenging activities (RSAs) of HAOA and its complex with Ga(III) may be due to a complex process, depending on the concentration, and on the environment, nature, and size of the free radical. The electron transfer pathway was considered as more probable in comparison to hydrogen donation in the scavenging of superoxide by 5-aminoorotic acid and its gallium(III) complex.

Biogenesis of HLA Ligand Presentation in Immune Cells Upon Activation Reveals Changes in Peptide Length Preference.

Induction of an effective tumor immunity is a complex process that includes the appropriate presentation of the tumor antigens, activation of specific T cells, and the elimination of malignant cells. Potent and efficient T cell activation is dependent on multiple factors, such as timely expression of co-stimulatory molecules, the differentiation state of professional antigen presenting cells (e.g., dendritic cells; DCs), the functionality of the antigen processing and presentation machinery (APPM), and the repertoire of HLA class I and II-bound peptides (termed immunopeptidome) presented to T cells. So far, how molecular perturbations underlying DCs maturation and differentiation affect the in vivo cross-presented HLA class I and II immunopeptidomes is largely unknown. Yet, this knowledge is crucial for further development of DC-based immunotherapy approaches. We applied a state-of-the-art sensitive MS-based immunopeptidomics approach to characterize the naturally presented HLA-I and -II immunopeptidomes eluted from autologous immune cells having distinct functional and biological states including CD14+ monocytes, immature DC (ImmDC) and mature DC (MaDC) monocyte-derived DCs and naive or activated T and B cells. We revealed a presentation of significantly longer HLA peptides upon activation that is HLA allotype specific. This was apparent in the self-peptidome upon cell activation and in the context of presentation of exogenously loaded antigens, suggesting that peptide length is an important feature with potential implications on the rational design of anti-cancer vaccines.

Azacytidine Sensitizes AML Cells for Effective Elimination By CD123 CAR T-Cells

Chimeric antigen receptor T-cells (CAR Tc) have yielded impressive remission rates in treatment-refractory B-cell malignancies (B-ALL and B-lymphomas) by targeting CD19, resulting in the first FDA approved CAR Tc therapies, Kymriah and Yescarta. However, the translation of these results for other cancer entities remains a challenge. Pre-clinical studies using second-generation CAR Tc against the interleukin-3 receptor alpha chain (CD123) engendered strong anti-leukemic activity. CD123 CAR Tc clinical studies resulted in transient responses, or complete remission but at the expense of on-target off-tumor toxicities. Our studies employing third-generation anti-CD123 CAR Tc demonstrate strong anti-leukemic activity with no adverse effects in vivo. However, the leukemia was not completely eradicated. Combining anti-CD123 CAR Tc with DNA hypomethylating (HMA) agents may enhance the anti-leukemic effect and survival. HMAs such as azacytidine (Aza) activate key epigenetically silenced pathways in AML cells, inhibiting cell proliferation while enhancing cell immunogenicity. We hypothesized that Aza will increase the expression of CD123 on AML cells resulting in long-term disease eradication by anti-CD123 CAR Tc. The anti-leukemic efficacy, survival advantage, safety and feasibility of the combination treatment with Aza and anti-CD123 CAR Tc were evaluated in vivo. HL-60 (CD123med), MLL-2 (CD123lo), MOLM-13 (CD123hi), primary de novo and relapsed/refractory (r/r) AML cells were cultured for 0-8 days in the presence of Aza (0µM-5µM) and analysed for their CD123 expression by flow cytometry, quantitative western blot and RNAseq. The anti-CD123 CAR was constructed with the humanized CSL362-based ScFv and the CD28-OX40-CD3ζ signaling domain, encoded in a third-generation lentiviral vector and expressed in CD3+ Tc from healthy donors. Rag2γc-/- mice (n=12-16/ group) were engrafted with 1x105 MOLM13/ffLuc AML cells and treated with PBS, 5x106 Non-transduced (NTD) Tc orCAR Tc, 4x 2.5mg/kg Aza, or 5x106 CAR Tc following 4x Aza (2.5mg/kg). Leukemic burden was assessed weekly by bioluminescence imaging. Tc activity and immunophenotyping was performed using flow cytometry at day 35 post engraftment, and survival was monitored. HL-60, MLL-2 and MOLM-13 cells showed significant increases in HLA-DR, PD-L1, STAT1 and IRF7 expression, as well as CD123 when exposed to Aza (Fig 1A,B). Interestingly, the increased effect was seen from day one regardless of concentration. This was similarly reflected in AML patient cells. Aza treatment also arrested cell proliferation and decreased viability in both cell lines and patient cells suggesting Aza can aid in the anti-leukemic effect. Rag2γc-/- mice engrafted with MOLM-13 and treated with Aza and CD123 CAR Tc demonstrated suppressed growth, and eradication of MOLM-13 cells compared to mice treated with CD123 CAR Tc or Aza alone. Additionally, a significant decrease in residual CD123+ cells in the bone marrow (BM) of dual treated mice was seen (Fig 1C). A higher frequency of residual CD8+ T-cells in the BM, and CD4+ Tc in the peripheral blood (PB) and BM of dual treated mice was observed compared to CAR Tc only treated mice. Most prominently, we found a significantly higher mean number of stem cell-like and central memory CD8+ Tc in the BM of dual treated mice (232 cells/µl and 208cells/µl, respectively) compared to the CAR Tc only group (55 cells/µl and 23 cells/µl, respectively). Assessment of immune checkpoint markers on residual CAR Tc of dual treated mice revealed significantly decreased levels of CTLA-4, PD-1 and TIM-3 in the BM, and CTLA-4 in the PB compared to the CAR Tc only group. While CAR Tc treatment alone demonstrated a survival advantage compared to PBS, NTD or Aza treated mice, Aza and CAR Tc treatment had a significantly higher survival rate compared to the CAR Tc only group (92% vs. 46% at day 50, p<.01). Our findings indicate that Aza increases immunogenicity and augments the cell surface expression of CD123 on AML cells, allowing enhanced recognition and elimination of malignant cells by CD123 CAR Tc. This is the first demonstration that HMAs and CAR Tc immunotherapy can be used synergistically to treat AML. Considering HMAs are currently under clinical investigation in AML, our data encourage further clinical evaluation of this dual treatment in r/r AML, including high-risk patients that are chemotherapy or allogeneic transplantation ineligible. Disclosures Hughes: Novartis, Bristol-Myers Squibb, Celgene: Research Funding; Novartis, Bristol-Myers Squibb: Consultancy, Other: Travel. White:BMS: Honoraria, Research Funding; AMGEN: Honoraria, Speakers Bureau. Yong:Novartis: Honoraria, Research Funding; Celgene: Research Funding; BMS: Honoraria, Research Funding.

Tumor-infiltrating mesenchymal stem cells: Drivers of the immunosuppressive tumor microenvironment in prostate cancer?

Prostate cancer is characterized by T-cell exclusion, which is consistent with their poor responses to immunotherapy. In addition, T-cells restricted to the adjacent stroma and benign areas are characterized by anergic and immunosuppressive phenotypes. In order for immunotherapies to produce robust anti-tumor responses in prostate cancer, this exclusion barrier and immunosuppressive microenvironment must first be overcome. We have previously identified mesenchymal stem cells (MSCs) in primary and metastatic human prostate cancer tissue. An Opal Multiplex immunofluorescence assay based on CD73, CD90, and CD105 staining was used to identify triple-labeled MSCs in human prostate cancer tissue. T-cell suppression assays and flow cytometry were used to demonstrate the immunosuppressive potential of primary MSCs expanded from human bone marrow and prostate cancer tissue from independent donors. Endogenous MSCs were confirmed to be present at sites of human prostate cancer. These prostate cancer-infiltrating MSCs suppress T-cell proliferation in a dose-dependent manner similar to their bone marrow-derived counterparts. Also similar to bone marrow-derived MSCs, prostate cancer-infiltrating MSCs upregulate expression of PD-L1 and PD-L2 on their cell surface in the presence of IFNγ and TNFα. Prostate cancer-infiltrating MSCs suppress T-cell proliferation similar to canonical bone marrow-derived MSCs, which have well-documented immunosuppressive properties with numerous effects on both innate and adaptive immune system function. Thus, we hypothesize that selective depletion of MSCs infiltrating sites of prostate cancer should restore immunologic recognition and elimination of malignant cells via broad re-activation of cytotoxic pro-inflammatory pathways.

Tertiary Lymphoid Structures in Colorectal Cancers and Their Prognostic Value.

BACKGROUND:Tumor-infiltrating lymphocytes (TIL) in tumour stroma are considered to be involved in the elimination of malignant cells and prevention of metastasis formation. TIL consist of T lymphocytes including cytotoxic lymphocytes that are a constituent part of the effector mechanism of anti-tumour immunity and B lymphocytes that can form tertiary lymphoid structures (TLS). TLS has been described in several solid tumours and colorectal carcinoma (CRC), and the influence on the local and systemic anti-cancer response.AIM:This study aimed to quantify the presence of TLS in CRC patients and to determine their role in tumour progression.PATIENTS AND METHODS:The study included 103 patients with CRC who underwent surgery at the University Clinic of Digestive Surgery in Skopje, whose operative material was analysed at the Institute of Pathology, Medical Faculty in Skopje. The density of TLS was determined and correlated with the neoplasm status of local growth (T), positive lymph nodes, lymphatic invasion, and stage of the disease and tumour grade.RESULTS:The density of TLS was significantly higher in patients with higher stage, lower T status, and negative lymph nodes, in patients with no lymphatic invasion and with better-differentiated tumours.CONCLUSION:The density of TLS plays an important role in controlling the tumour growth, and it can be a parameter for neoplasm progression in CRC patients. The density of TLS influences the control of tumour progression.

Dendritic Cell Production From Allogeneic Donor CD34+ Stem Cells and Mononuclear Cells for Patients With AML: Cancer Vaccine

Background: Active immunotherapy provides better recognition of tumor-related antigens by immune system of patient, enhanced immune system and elimination of malignant cells. This modality employs therapeutic potential of donor specific and tumor specific immune responses. Active immunotherapy targets immunosuppressive and tolerogenic mechanisms suppressed by tumor cells. Aim: T lymphocytes and antigen-presenting cells (dendritic cells) are 2 cell lineages that play crucial role in the battle of organism against cancer. Close similarity between cancer cells and normal cell structure is the most important reason of escape from defense cells, namely T lymphocytes. Stimulation and enhancement of T lymphocytes against cancer cells comprise principal part of therapy. Methods: To generate allogeneic dendritic cells, leukemic stem cells were isolated from bone marrow samples from patients with acute leukemia. Lysate was prepared from leukemic stem cells identified by flow cytometer. Stem cells and mononuclear cells (1 × 10 > 6/kg) obtained from sibling donors by apheresis were separated to produce dendritic cells. For dendritic cell transformation, GM-CSF and IL-4 were added to media where leukemic stem cell lysate from patient and mononuclear cells from sibling donor were treated. From samples taken from the culture medium, after 48, 72 and 96 hours, dendritic cell surface markers (CD80, CD83 and CD86) was assessed by flow cytometry. CD3, CD14, CD19, CD56, CD66b-negative, and HLA-DR, CD86 positive are indicative of immature DC. HLA-DR, CD80 and CD83 positive are indicative of mature DC. Results: Mononuclear cells were detected by 27% among allogeneic hematopoietic cell groups harvested by apheresis. After culture under GMP conditions, mononuclear cell rate was found to be 24% on hours 96 and 120. It was seen that 88% of mononuclear cells transformed to mature dendritic cells after 96 hours culture. Conclusion: In cancer patients, minimal residual disease can be eliminated by active tumor vaccine after reducing tumor burden by standard methods. Tumor vaccine obtained from allogeneic sibling donor can be used in lieu of autologous tumor vaccine and it is thought to be more effective. Allogeneic dendritic cells produced at 37°C in CO2 media under GMP conditions can be used in tumor immunotherapy. More effective method would have been used by employing dendritic cells against cancer stem cells rather than cancer cells itself.

Interaction Between Cap- Derived Singlet Oxygen And Tumor Cell Protective Catalase: Update And Chances

Transformation of cells from various tissues requires NADPH oxidase-dependent generation of extracellular superoxide anions. These drive the proliferation, but also cause the elimination of malignant cells through the HOCl and the NO/peroxynitrite signaling pathways. These intercellular signaling pathways induce apoptosis selectively in malignant cells, due to site-specific concerted interaction of defined reactive oxygen and nitrogen species (ROS/RNS). Tumor progression requires the expression of membrane-associated catalase. This enzyme interferes with HOCl signaling through decomposition of H2O2, and with NO/peroxynitrite signaling through oxidation of NO and decomposition of peroxynitrite. Membrane-associated catalase has been found on all lines of bona fide tumor cells and represents a promising target for novel antitumor strategies. Inactivation of tumor cell-specific membrane-associated catalase reactivates intercellular ROS/RNS-dependent apoptosis-inducing signaling and leads to autocrine apoptotic selfdestruction of tumor cells. Model experiments with defined ROS and RNS led to the conclusion that CAP-derived singlet oxygen might lead to site-specific inactivation of catalase, followed by tumor cell-specific generation of secondary singlet oxygen and further inactivation of catalase. This then allows for subsequent reactivation of intercellular ROS/RNS-dependent apoptosis-inducing signaling [1]. Only on the first sight, this model seemed a) to be in contradiction to the model on the dependence of CAP action from aquaporins [2] and b) to be independent of subsequent immunogenic cell death and activation of a cytotoxic T cell response [3, 4]. The analysis of existing experimental data from several groups and the alignment of site-specific mechanisms, defined by chemical biology and cell biology, allowed to establish an updated and comprehensive model [5] that includes the concepts from references [1-4] in a rational way. This model shows several biochemical amplification loops related to the generation of secondary singlet oxygen, a positive feed-back of HOCl signaling on immunogenic modulation, as well as a feedback loop from activated T cells to catalase inactivation and reactivation of intercellular ROS/RNS signaling. Thereby HOCl seems to play a role as mediator for apoptosis induction and enhancer of immunogenic stimulation. The detailed biochemical analysis of generation of secondary singlet oxygen by tumor cells after initial interaction with exogenous singlet oxygen allowed to predict that an increase in the concentrations of tumor cell-derived extracellular superoxide anions and/or nitric oxide should cause a substantial synergistic effect with exogenous singlet oxygen. Therefore, a hybrid molecule, consisting of a plant-derived NOX stimulator and a plant-derived inhibitor of NO dioxygenase (NOD) (patent application PCT/EP2016/080169) has been synthesized in collaboration with Prof. R. Bruckner and Dr. S. Braukmuller (Organic Chemistry, Freiburg). When applied in vitro at 0.1-0.5 nmoles/liter, this compound caused a remarkable synergistic effect with exogenous singlet oxygen, characterized by a reduction of the optimal dose of singlet oxygen by three log steps. We suggest to evaluate whether this principle can be translated into application of CAP for tumor treatment. If so, the reduction of necessary plasma doses might be useful for further development of microinvasive plasma-mediated tumor therapy

Dendritic Cell Production from Allogeneic Donor Cd34+ Stem Cells and Mononuclear Cells; Cancer Vaccine

Aim: Active immunotherapy provides better recognition of tumor-related antigens by immune system of patient, enhanced immune system and elimination of malignant cells. This modality employs therapeutic potential of donor specific and tumor specific immune responses. Active immunotherapy targets immunosuppressive and tolerogenic mechanisms suppressed by tumor cells. T lymphocytes and antigen-presenting cells (dendritic cells) are two cell lineages that play crucial role in the battle of organism against cancer. Close similarity between cancer cells and normal cell structure is the most important reason of escape from defense cells, namely T lymphocytes. Stimulation and enhancement of T lymphocytes against cancer cells comprise principal part of therapy.Material and Method: To generate allogeneic dendritic cells, leukemic stem cells were isolated from bone marrow samples from patients with acute leukemia. Lysate was prepared from leukemic stem cells identified by flow cytometer. Stem cells and mononuclear cells (1x10>6/kg) obtained from sibling donors by apheresis were separated to produce Dendritic Cells. For Dendritic Cell transformation, GM-CSF and IL-4 were added to media where leukemic stem cell lysate from patient and mononuclear cells from sibling donor were treated. From samples taken from the culture medium, after 48, 72 and 96 hours, dendritic cell surface markers (CD 80, CD 83 and CD 86) was assessed by flow cytometry. CD3, CD14, CD19, CD56, CD66b-negative, and HLA-DR, CD86 positive are indicative of immature DC. HLA-DR, CD 80 and CD83 positive are indicative of mature DC.Results: Mononuclear cells were detected by 27 % among allogeneic hematopoietic cell groups harvested by apheresis. After culture under GMP conditions, mononuclear cell rate was found to be 24% on hours 96 and 120. It was seen that 88% of mononuclear cells transformed to mature dendritic cells after 96 hours culture.Conclusion: In cancer patients, minimal residual disease can be eliminated by active tumor vaccine after reducing tumor burden by standard methods. Tumor vaccine obtained from allogeneic sibling donor can be used in lieu of autologous tumor vaccine and it is thought to be more effective. Allogeneic dendritic cells produced at 37¡C in CO2 media under GMP conditions can be used in tumor immunotherapy. More effective method would have been used by employing dendritic cells against cancer stem cells rather than cancer cells itself. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

CD7 CAR for the Treatment of Acute Myeloid and Lymphoid Leukemia

CD7 is a T- and NK-cell marker highly expressed in T cell leukemia and in 30% of AML cases, where its presence correlates with chemoresistance and worse prognosis. We created CD7-directed chimeric antigen receptors (CARs) using single chain variable fragment (scFv) sequences derived from three different CD7-specific mAb clones and evaluated the feasibility and potency of using CD7 CAR T cells for the treatment of the CD7+ hematologic malignancies. Since normal T cells themselves express CD7, their transduction with CD7 CARs limited their expansion in vitro, resulting in approximately 30-fold fewer T cells after 14 days of culture compared to control CAR-transduced cells. To circumvent this problem of fratricide, we used the CRISPR/Cas9 system to disrupt the CD7 gene in T cells prior to retroviral transduction with CD7 CAR. The CD7 gene was successfully disrupted in 90% of T cells, minimizing fratricide of CD7 CAR T cells, and leading to a robust T cell expansion, similar to that of control T cells transduced with an irrelevant CAR (310-fold vs 380-fold expansion after 14 days, respectively). Lack of CD7 did not compromise CAR T cell effector function, as we observed equal cytotoxicity and expansion in CD7+ and CD7- CD19 CAR T cells upon coculture with a CD19+ cell line Raji (P=0.99 and P=0.86, respectively).Expanded CD7-deficient CD7 CAR T cells demonstrated robust cytotoxicity against CD7+ AML cell lines KG-1a and Kasumi-3 and T-ALL cell line CCRF-CEM, resulting on average in a 19-fold expansion of CAR T cells and elimination of malignant cells (92-99.9%) after 7 days of co-culture at a 1:4 initial effector-to-target ratio. This cytotoxicity was CD7-specific, as CD7 CAR T cells showed no activity against a CD7-negative cell line Raji. We also observed potent activity of CD7 CAR T cells against primary AML cells, leading to an 80% reduction in blast counts after 48 hours of co-culture at a 1:1 effector-to-target ratio, compared with control CAR T cells. Moreover, a 5-hour co-culture of CD7 CAR T cells with primary AML blasts and subsequent culture on a methylcellulose medium for 12 days resulted on average in a 26-fold reduction in leukemic colonies; these data suggest that the CD7 CAR T cells can eliminate primitive leukemic progenitors.Because CD7 is expressed on some myeloid progenitors, we assessed the activity of CD7 CAR T cells against normal myeloid precursors. After coculturing cord blood cells with CD7 CAR T cells at a 10:1 effector-to-target ratio and subsequently expanding myeloid colonies on a methylcellulose medium for 14 days, we observed no significant differences in the numbers of monocytic, erythrocytic and granulocytic colonies compared to a coculture with control CAR T cells. Hence, CD7 CAR T cells appear non-toxic to normal myeloid progenitor cells.In summary, we show that CD7 CAR T cells can be efficiently generated and expanded following genomic disruption of the CD7 gene by CRISPR/Cas9. Expanded CD7 CAR T cells produce robust cytotoxic activity against AML and T-ALL cell lines as well as primary AML blasts, and show no toxicity against normal myeloid progenitors. These results demonstrate the potency and support the feasibility of using CD7 CAR T cells for the targeted therapy of acute myeloid and lymphoid leukemia. DisclosuresBrenner:Viracyte: Equity Ownership; Cell Medica: Patents & Royalties.

Abstract 3505: A bioluminescent, homogeneous annexin V microplate-based method for assessment of apoptosis

Abstract The selective elimination of malignant cells via the apoptotic process continues to be the cornerstone of modern anti-cancer therapy regimens. Therefore, in vitro screening approaches aimed at identifying clinically useful apoptosis inducers remain critically important. Recently, phenotypic screening methods have enjoyed a resurgence due to more biologically complex and relevant cell models as well as advances in chemical proteomics which have allowed for more successful target identification. As a consequence, novel probes and tools with enabling attributes are required to fully realize this discovery potential. In an effort to address this unmet need, we have developed a bioluminescent and homogeneous annexin V binding assay for the assessment of apoptosis. Unlike traditional fluorescent annexin V methodology, the “no-wash” reagent employed in this new assay utilizes binary components of a novel luciferase separately fused to annexin V. The annexin V-luciferase subunit fusion pairs have low intrinsic affinity for each other and thus produce no or low luminescence until phosphatidylserine (PtdSer) exposure drives annexin-fusion pair oligimerization. Ultimately, this protein:protein interaction on or near the cell surface reconstitutes full luciferase activity causing an increase in luminescence in the presence of a luciferase substrate. A separate, pro-fluorescent, multiplexed component of the reagent further delineates differences in annexin positivity based on maintenance or loss of membrane integrity corresponding to apoptosis or necrosis, respectively. We validated this method using a panel of diverse cancer cell lines (U2-OS, DLD-1, HeLa, Jurkat, K562, A549, and PC-3), representing both attachment-dependent and -independent morphologies after dose-dependent challenge with intrinsic (bortezomib, panobinostat, staurosporine, and paclitaxel) and extrinsic (rhTRAIL) inducers of apoptosis as well as agents known to produce primary necrosis (ionomycin and digitonin). Caspase activation data was also collected in parallel plates at endpoint as a well-validated and sensitive orthogonal comparator. The bioluminescent annexin V method proved sufficiently robust in 384 well microplate formats to routinely produce Z’ > 0.7 and rank-order potencies in good agreement with caspase activation values. In addition to this microplate functionality, the reagent allowed for sensitive, facile imaging of apoptotic induction in living cells using different imaging platforms. Taken together, the method and reagent should provide unparalleled flexibility with regard to live cell apoptosis detection in both conventional microplate and high content-like imaging formats and advance the pace of new chemical entity discovery. Citation Format: Kevin Kupcho, John Shultz, Andrew Niles, Wenhui Zhou, Robin Hurst, Jim Hartnett, Thomas Machleidt, Terry Riss, Dan Lazar, Jim Cali. A bioluminescent, homogeneous annexin V microplate-based method for assessment of apoptosis. [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 3505.

Harnessing the apoptotic programs in cancer stem-like cells.

Elimination of malignant cells is an unmet challenge for most human cancer types even with therapies targeting specific driver mutations. Therefore, a multi-pronged strategy to alter cancer cell biology on multiple levels is increasingly recognized as essential for cancer cure. One such aspect of cancer cell biology is the relative apoptosis resistance of tumor-initiating cells. Here, we provide an overview of the mechanisms affecting the apoptotic process in tumor cells emphasizing the differences in the tumor-initiating or stem-like cells of cancer. Further, we summarize efforts to exploit these differences to design therapies targeting that important cancer cell population.

Resistance of Hematopoietic Stem Cells Lacking the KIR Ligand to Autologous NK Cell Attacks in Patients with Acquired Aplastic Anemia

Normal blood cells, including hematopoietic stem cells (HSCs), express KIR ligands (KIR-Ls) to protect themselves from an autologous NK cell attack, and malignant cells lacking KIR-Ls elicit NK cell-mediated killing of themselves. This missing-self mechanism is believed to play an important role in the elimination of malignant cells. However, the mechanisms underlying the killing of KIR-L-lacking malignant cells by NK cells remain unclear due to the heterogeneity of tumor cells in terms of their proliferative capacity, and also because other accessory molecules may be involved in the NK cell attacks, in addition to KIR-Ls. This makes it difficult to clarify the interaction between NK cells and KIR-L-lacking target cells. The lack of class I HLA occurs not only in malignant blood cells, but also in the normal leukocytes of patients with acquired aplastic anemia (AA). These HLA-lacking leukocytes, detectable in 13% of patients with AA, are derived from HSCs that undergo copy number neutral loss of heterozygosity of the short arm of chromosome 6 (6pLOH), and thereby escape the cytotoxic T-cell (CTL) attack against HSCs. The 6pLOH may involve KIR-L loss in some patients, leading to a change in the susceptibility of the affected HSCs to NK cell-mediated killing. Unlike malignant cells, HLA-lacking leukocytes are essentially the same as the wild-type leukocytes, except for the HLA expression. Studying 6pLOH (+) AA patients with leukocytes lacking KIR-Ls should therefore be useful for clarifying the roles of KIR-Ls and other accessory molecules in the target cell killing by NK cells. Screening of 389 patients with AA using flow cytometry and a SNP array analysis revealed that there were HLA-A allele-lacking leukocytes in 60 (15.4%) patients, which included 36 C1/C2 and 24 Bw4/Bw6 heterozygotes. Unexpectedly, a lack of KIR-Ls as a result of 6pUPD was found in five patients (13.9%, C1 missing in two and C2 missing in three) of the 36 C1/C2 heterozygotes and in five (20.8%) of the 24 Bw4/Bw6 heterozygotes, although the proportion of patients lacking a KIR-L-containing haplotype (20.8%) was significantly lower than that of patients lacking a haplotype that did not contain KIR-Ls (79.2%). Moreover, the median percentage of HLA-A-lacking granulocytes in the 10 patients who lacked a KIR-L-containing haplotype (12.4%, 0.44%-50.3%) was significantly lower than that (55.3%, 1.4%-99.4%) in the 26 patients who lacked a haplotype that did not contain KIR-Ls, suggesting that the HSCs lacking KIR-Ls or their progenies are susceptible to autologous NK cells to some extent, but are not eliminated completely. To clarify the mechanisms underlying the HSC resistance to NK cells, we determined the KIR gene repertoire and the haplotype of seven patients whose 6pLOH(+) leukocytes were lacking a KIR-L-containing haplotype. All patients possessed inhibitory KIR genes responsive to corresponding KIR-Ls, a finding that negates the possibility that NK cells failed to undergo licensing in these patients. Although the frequency of the KIR-B haplotype, a haplotype associated with a higher cytotoxic function of NK cells, in the seven patients was lower (14%) than that in Japanese healthy individuals (40.1%), two patients possessed the KIR-B haplotype. Phenotypic analyses of the NK cell subsets defined by anti-2DL1, anti-2DL2/2DL3 and anti-3DL1 antibodies showed that all seven patients had a similar percentage of the eight different NK cell subsets, which included 0.5 to 8% of effector NK cells capable of killing leukocytes lacking corresponding KIR-Ls. The expression level of HLA-E was comparable between HLA-A-lacking and HLA-A-retaining monocytes. The expression of NKG2A on the effector NK cells was also comparable to that of the other NK cell subsets in the 6pLOH(+) patients. Our study demonstrated, for the first time, that HSCs lacking KIR-Ls can evade autologous NK cell attack through an as yet unknown mechanism(s) and can continue to generate blood cells in patients with AA. DisclosuresNo relevant conflicts of interest to declare.

Oxidative Stress Reduced by a Green Tea Concentrate and Capsicum Combination: Synergistic Effects

ABSTRACTReactive oxygen species that are produced by aerobic metabolism and signaling cascades have the potential to play important roles in maintaining homeostatic redox and cell proliferation. When the balance between the production and elimination of reactive oxygen species is perturbed toward production, the result is oxidative stress. High levels of oxidative stress are a general characteristic of cancer. The altered redox state within a tumor microenvironment confers a growth advantage through increased proliferation rates, evasion of apoptosis, and increased resistance to therapeutic compounds. We have tested a synergistic combination of green tea—Camellia sinensis—concentrate and powdered Capsicum powder (TeaFense™/Capsol-T™) as a dietary supplement to reduce oxidative stress as an approach to elimination of malignant cells. Here, we demonstrate that the green tea-powdered Capsicum mixture effectively reduces levels of oxidative stress in both cancer (HeLa) and noncancer (MCF-10A) cells as determined from measurements of levels of the oxidative stress indicator Nrf-2 by western blot analysis. Nrf-2 is a transcription factor that controls an antioxidant response element. Increased expression of Nrf-2 is linked to high levels of oxidative stress and vice versa. Based on levels of Nrf-2, the mixture of green tea concentrate plus powdered Capsicum reduced oxidative stress by more than 50% compared with 15% by the green tea concentrate alone.

1,000 Ways to die: natural compounds modulate non-canonical cell death pathways in cancer cells

Over the past decades, apoptosis emerged as a leading strategy to eliminate cancer cells. Nowadays, it becomes progressively clear that apoptosis is not the only programmed cell death mechanism implicated in the elimination of malignant cells. This review will discuss non-apoptotic cell death modalities and will focus on natural compounds acting as inducers of these recently described processes. We will describe the major molecular characteristics of each individual death mechanism and we will detail the effects of natural and naturally-derived compounds on these cellular mechanisms.

Coordinated regulation of the immunoproteasome subunits by PML/RARα and PU.1 in acute promyelocytic leukemia

Recognition and elimination of malignant cells by cytotoxic T lymphocytes depends on antigenic peptides generated by proteasomes. It has been established that impairment of the immunoproteasome subunits, that is, PSMB8, PSMB9 and PSMB10 (PSMBs), is critical for malignant cells to escape immune recognition. We report here the regulatory mechanism of the repression of PU.1-dependent activation of PSMBs by PML/RARα in the pathogenesis of acute promyelocytic leukemia (APL) and the unidentified function of all-trans retinoic acid (ATRA) as an immunomodulator in the treatment of APL. Chromatin immunoprecipitation and luciferase reporter assays showed that PU.1 directly bound to and coordinately transactivated the promoters of PSMBs, indicating that PSMBs were transcriptional targets of PU.1 and PU.1 regulated their basal expression. Analysis of expression profiling data from a large population of acute myeloid leukemia (AML) patients revealed that the expression levels of PSMBs were significantly lower in APL patients than in non-APL AML patients. Further evidence demonstrated that the decrease in their expression was achieved through PML/RARα-mediated repression of both PU.1-dependent transactivation and PU.1 expression. Moreover, ATRA but not arsenic trioxide induced the expression of PSMBs in APL cells, indicating that ATRA treatment might activate the antigen-processing/presentation machinery. Finally, the above observations were confirmed in primary APL samples. Collectively, our data demonstrate that PML/RARα suppresses PU.1-dependent activation of the immunosubunits, which may facilitate the escape of APL cells from immune surveillance in leukemia development, and ATRA treatment is able to reactivate their expression, which would promote more efficient T-cell-mediated recognition in the treatment.