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Supplementary Figure 1 from Crucial Roles for Protein Kinase C Isoforms in Tumor-Specific Killing by Apoptin

The chicken anemia virus–derived protein apoptin induces apoptosis in a variety of human malignant and transformed cells but not in normal cells. However, the mechanisms through which apoptin achieves its selective killing effects are not well understood. We developed a lentiviral vector encoding a green fluorescent protein–apoptin fusion gene (LV-GFP-AP) that can efficiently deliver apoptin into hematopoietic cells. Apoptin selectively killed the human multiple myeloma cell lines MM1.R and MM1.S, and the leukemia cell lines K562, HL60, U937, KG1, and NB4. In contrast, normal CD34+ cells were not killed and maintained their differentiation potential in multilineage colony formation assays. In addition, dexamethasone-resistant MM1.R cells were found to be more susceptible to apoptin-induced cell death than the parental matched MM1.S cells. Death susceptibility correlated with increased phosphorylation and activation of the apoptin protein in MM1.R cells. Expression array profiling identified differential kinase profiles between MM1.R and MM1.S cells. Among these kinases, protein kinase Cβ (PKCβ) was found by immunoprecipitation and in vitro kinase studies to be a candidate kinase responsible for apoptin phosphorylation. Indeed, shRNA knockdown or drug-mediated inhibition of PKCβ significantly reduced apoptin phosphorylation. Furthermore, apoptin-mediated cell death proceeded through the upregulation of PKCβ, activation of caspase-9/3, cleavage of the PKCδ catalytic domain, and downregulation of the MERTK and AKT kinases. Collectively, these results elucidate a novel pathway for apoptin activation involving PKCβ and PKCδ. Further, they highlight the potential of apoptin and its cellular regulators to purge bone marrow used in autologous transplantation for multiple myeloma. Cancer Res; 70(18); 7242–52. ©2010 AACR.

Supplementary Figure 2 from Crucial Roles for Protein Kinase C Isoforms in Tumor-Specific Killing by Apoptin

Supplementary Table 1 from Crucial Roles for Protein Kinase C Isoforms in Tumor-Specific Killing by Apoptin

Supplementary Methods from Crucial Roles for Protein Kinase C Isoforms in Tumor-Specific Killing by Apoptin

Supplementary Figure 3 from Crucial Roles for Protein Kinase C Isoforms in Tumor-Specific Killing by Apoptin

Supplementary Table 3 from Crucial Roles for Protein Kinase C Isoforms in Tumor-Specific Killing by Apoptin

Disease remission is successfully induced in the majority of patients with acute myeloid leukemia (AML) or myelodysplastic syndrome (MDS), but is generally not sustained without stem cell transplantation. Adoptive immunotherapy offers an attractive option to consolidate AML remission. Here, we hypothesized that ex-vivo expanded Vg9Vd2 T-cells will mediate a therapeutically beneficial graft versus leukemia (GvL) effect in this disease. Peripheral blood mononuclear cells from healthy donors and from patients with AML were activated in-vitro using zoledronic acid and a cytokine cocktail, containing IL-2. As a result, we observed an average 200-fold expansion of Vg9Vd2 T-cells from AML patients, over 15 days. Such expansion was only observed in patients without circulating AML blasts. Expanded patient-derived Vg9Vd2 T-cells exhibited a less differentiated phenotype than cells from healthy donors, indicated by higher expression of CD62L, CCR7 and CD27. A four hour Annexin V cytotoxicity assay using KG-1 and U937 leukemic cells showed that these Vg9Vd2 T-cells were cytotoxic and produced nanogram amounts of interferon-g; (IFN-g). In a parallel approach, we investigated feasibility of developing a universal allogeneic Vg9Vd2 T-cell therapy. Feasibility of this approach has been demonstrated in a small trial in which haploidentical donor-derived Vg9Vd2 T-cells were safely infused and achieved an efficacy signal in patients with advanced haematological diseases. We have recently developed a novel method to expand Vg9Vd2 T-cells from healthy donors ex-vivo (“Method 2 (M2)” cells – patent protected), which improves their expansion over 2 weeks to 1700 fold. Cytotoxicity data shows that M2 cells destroy luciferase-expressing U937 and KG-1 target cells at 2-3 fold enhanced efficiency, accompanied by 2-3.5-fold increased IFN-g; release when compared to cells expanded using conventional approaches. Furthermore, M2 cells can achieve serial killing of AML cell lines through up to 4 cycles of re-stimulation. Treatment of SCID-Beige mice with an established U937 leukemic burden is also more effective using M2 expanded Vg9Vd2 T-cells, particularly when combined with zoledronic acid. Together these data highlight the promise of Vg9Vd2 T-cell immunotherapy of AML. Citation Format: Ana C. Parente-Pereira, Lynsey M. Whilding, Pramila Krishnamurthy, Richard Beatson, Tomasz Zabinski, Linda Barber, Farzin Farzaneh, Ghulam Mufti, John Maher. Immunotherapy of acute myeloid leukemia using Vg9Vd2 T-cells. [abstract]. In: Proceedings of the CRI-CIMT-EATI-AACR Inaugural International Cancer Immunotherapy Conference: Translating Science into Survival; September 16-19, 2015; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(1 Suppl):Abstract nr B124.

In general, ‘poorly’ differentiated tumours have a worse prognosis when compared to more ‘well’ differentiated ones. Therefore, the use of a biological agent that could promote differentiation might have a therapeutic advantage. CCAAT enhancer binding protein alpha (C/EBPα) is a transcription factor known to be involved in the regulation of cell differentiation in a number of tissue types. Loss of C/EBPα expression, for example, causes abnormal levels of biliary transcription factors, impaired hepatocyte maturation and liver fibrosis. In contrast C/EBPα overexpression inhibits the development of hepatocellular carcinoma, restores myeloid differentiation and prevents hyperproliferation of hematopoietic cells in acute myeloid leukemia. In this study, we tested the effect of stimulation of C/EBPα expression by a specific small activating RNA (saRNA) on a panel of cell lines representing both well-differentiated and poorly-differentiated cancer cell types. The C/EBPα-saRNA inhibited proliferation of poorly differentiated small cell lung cancer and pancreatic cancer cell lines compared to treatment with scrambled double-stranded RNA controls. However C/EBPα-saRNA was not as effective in suppressing proliferation in well-differentiated insulinoma and breast cancer (MCF7) cell lines. Comparison of endogenous levels of C/EBPα, using qPCR and Western blots, showed that undifferentiated tumour cell lines expressed lower levels of C/EBPα when compared to the well-differentiated tumor types. Gene expression analysis in tumours from xenografts and cirrhotic DEN treated HCC rats, demonstrated that saRNA mediated stimulation of C/EBPα expression, could suppress the steady-state transcript levels for KLF4, OCT3, SOX2, C-Myc and C-Kit. The inhibited expression of these transcription factors correlated with greater expression of differentiation markers and reduced epithelial mesenchymal transition by the C/EBPα saRNA treated cells. Our results suggest that saRNA mediated stimulation of C/EBPα, could be of potential therapeutic value, especially in poorly differentiated cancers. Furthermore, intracellular expression levels of C/EBPα could be an important prognostic factor for predicting the therapeutic response in poor or un-differentiated tumors. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):B225. Citation Format: K W. Huang, Vikash Reebye, Paul J. Mintz, Pal Saetrom, Piotr Swiderski, L Peng, C Liu, X X. Liu, Steen Lindkaer Jensen, Dimitris Zacharoulis, Nikos Kostomitsopoulos, Noriyaki Kasahara, Joanna Nicholls, Long R. Jiao, Madhava Pai, Duncan Spalding, Farzin Farzaneh, Malkhaz Mizandari, Tina Chikovani, Mohammed Emara, Abdelali Haoudi, Don Tomalia, John J. Rossi, Nagy A. Habib. Short activating RNA (saRNA) targeting C/EBPA significantly inhibits cell proliferation of undifferentiated cancer cells. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr B225.

Background: One component of the therapeutic efficacy of IgG1 monoclonal antibodies (mAb) is their ability to induce antibody dependent cellular cytotoxicity (ADCC) by NK cells and other immune effectors bearing the low-affinity Fc RIIIa (CD16) receptor. Clinical studies establish the relevance of ADCC finding widespread polymorphisms that encode a very low affinity CD16 (158F/F) predict limited clinical responses to Cetuximab or Rituximab. Now, mAbs with increased affinity for CD16 can be produced using GlycoMab technology. The altered glycosylation pattern in the Fc domain of these antibodies enhances CD16-binding and ADCC irrespective of genotype. Yet, the potential for enhanced ADCC in patients with advanced colorectal carcinoma (CRC) has not been demonstrated. Considering the potential for tumor-associated immune impairments and treatments-associated leukopenia, we enumerated and characterized NK cells from patients prior to chemotherapy, on active therapy, or following two-lines of standard treatment and compared responsiveness to standard and glycoengineered anti-EGFR mAbs. Methodology: PBMCs were isolated from blood of age-matched healthy donors and 100 patients: either at presentation with metastatic disease; during chemotherapy with FOLFOX, FOLFIRI, or Irinotecan; or at disease progression >4 weeks following second line failure. Laboratory tests included: immunophenotyping for CD3,CD8,CD45,CD4 and CD3,CD56/16,CD45,CD19 markers; measurements of CD16- and NKG2D-expressing NK cells; CD16 158 genotype; and assessment of K562-killing (natural cytotoxicity) and degranulation (CD107+) of NK cells among cryopreserved PBMC in response to K562 or EGFR+ A431 cells with 10 g/ml GlycoMAb anti-EGFR (GA201), wild-type GA201, Panitumumab or Cetuximab. Results: For each group of patients, the proportion of NK cells among lymphocytes remained similar to controls. However, NK cells counts were reduced about half in patients (affected primarily by disease and only slightly by treatment). Per NK cell killing capacity of K562 was substantially impaired in patients prior to therapy and most profoundly among the post-chemotherapy group. ADCC responses were similarly impaired but to lesser degrees. In all cohorts tested, GlycoMAb anti-EGFR was superior to cetuximab in activating more NK cells. The superior ADCC activation of NK cells by GlycoMAb was most pronounced in the patients with chemotherapy-resistant disease. Conclusion: Chemotherapy does not induce gross alterations in the immune phenotype of CRC patients. At all stages of treatment, patients retain CD16+ NK cells capable of ADCC. The impairments in the NK functional responses tested are likely due to host/tumor interaction and not a consequence of treatment. Despite impairments, the best activation of NK cells was always in response to GlycoMAb GA201 for all individuals tested. These findings support testing for enhanced ADCC by GA201 in clinical trials of CRC patients following two lines of chemotherapy or in combination with chemotherapy. Acknowledgements: Simon Hollingsworth, current affiliation Astra Zeneca UK Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr C221.