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Z. Zhang



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    P2.04 - Poster Session 2 - Tumor Immunology (ID 154)

    • Event: WCLC 2013
    • Type: Poster Session
    • Track: Biology
    • Presentations: 1
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      P2.04-003 - Novel mechanism of immune-tolerance due to aberrant expression of Natural Killer-Cell Immunoglobulin-like Receptors (KIRs) and enhanced platelet interactions (ID 3389)

      11:30 - 12:30  |  Author(s): Z. Zhang

      • Abstract

      Background
      Cancer metastasis is the main cause of cancer-related deaths. Metastatic cancer cells spread through blood vessels where they constantly interact with platelets and leukocytes, forming tumor microemboli and thereby protected from otherwise rapid elimination from host immune defense cells such as NK cells. Platelets are known to be prometastatic and pro-angiogenic. They release platelet-derived growth factors, chemokines, various adhesion molecules and coagulation factors that effectively promote cancer spreading and metastasis. We previously demonstrated that many metastatic cancer cells acquire immune-resistance by aberrant expression of KIRs on their surface. We showed that cancer cells with aberrant expression or with forced ectopic expression of KIRs are more resistant to NK killing than those with null or low KIR expression. Pre-blocking with anti-KIR antibodies effectively reverses their sensitivity to NK killing. Here we report that KIR-expressing cancer cells interact strongly with platelets leading to increase NK tolerance compared to cancer cells with null or low KIR expression. These data support a novel mechanism of immune-tolerance due to aberrant KIR expression on metastatic cancer cells.

      Methods
      DNA microarrays were performed on metastatic lung cancer cells re-derived from orthotopic human metastatic lung tumors in athymic nude rats. Aberrant expression of KIR genes was detected and verified with immunohistochemistry and flow cytometry. For ectopic expression, lung adenocarcinoma parental cells (H2122-GFP) were transfected with KIR2DL1 (LL454) and/or KIR3DL1 (LL456) plasmids. Stable transformants were enriched by cell sorting. Binding of these cancer cells with differential KIR expression with human platelets, pre-labeled with anti-CD41-APC antibodies, were analyzed by flow cytometry. NK killing of GFP-tagged cancer cells with differential KIR expressions in the presence and absence of human platelets was accessed with fluorescence intensity in a fluorescent plate reader.

      Results
      Using in vitro cytotoxic assays, we found that KIR expression or platelet coating on cancers clearly increased their resistance to NK cell killing when compared with parental cells. Interestingly, we found that platelet coating on those metastatic cancer cells with high aberrant KIR expression increased their IC50 values by 6 to 14 folds respectively when compared with parental cells, while platelet coating on those cells with forced KIR expression also increased their IC50 values by 9 to 12 folds respectively. Our observation shows that NK tolerance correlates positively with platelet coating and the levels of KIR expression on the cancer cells (with correlation coefficient = 0.9 to 0.98), and that the NK resistance is the highest when both KIR aberrant expression and platelet coating are present on the cancer cells.

      Conclusion
      We discovered a novel mechanism of immune resistance to NK cells due to aberrant expression of KIRs on cancer cells. Aberrant KIR expression on cancer cells enhanced their interaction with platelets leading to further increase in NK tolerance than those cells with null or low KIR expression. This observation suggests that anti-platelet antagonists and anti-KIR antibodies may have clinical potential for the treatment or prevention of metastatic and immune resistant cells.

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    P2.05 - Poster Session 2 - Preclinical Models of Therapeutics/Imaging (ID 158)

    • Event: WCLC 2013
    • Type: Poster Session
    • Track: Biology
    • Presentations: 1
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      P2.05-017 - Evaluation of the selective aurora B kinase inhibitor AZD1152 in SCLC lines with and without MYC family amplification. (ID 2877)

      09:30 - 09:30  |  Author(s): Z. Zhang

      • Abstract

      Background
      Background: Small cell lung cancer (SCLC) has an overall 5-year survival rate of < 5% despite initial response rates to first line chemotherapy of 70-90%. MYC family amplification occurs in 30% of SCLC and MYC amplified tumors have been shown to be dependent on aurora kinase B for their survival. Aurora B kinase (AURKB) is a chromosomal passenger protein that plays a critical role in mitosis by regulating chromosome alignment, accurate segregation, and cytokinesis during the mitotic stages. We evaluated the effects of the selective aurora B kinase inhibitor AZD1152-HQPA (active drug) in a panel of 15-SCLC lines.

      Methods
      Methods: Nine lines had MYC-family amplification (MYC-5 lines, MYCL1-2 lines, MYCN-2 lines) and 6-lines had no MYC-family amplification. Growth inhibition by AZD1152-HQPA was assessed by tetrazolium based assays. Apoptosis was determined using the DNA binding dyes YOPRO and propidium iodine (PI) and analysis by flow cytomery. The induction of polyploidy was evaluated by flow cytometry following PI staining of the DNA. The effect of AZD1152-HQPA on anchorage independent growth was determined by soft agar colony forming assays. Finally, we evaluated the in vivo efficacy of AZD1152 (prodrug) on SCLC xenografts in nude mice.

      Results
      Results: AZD1152-HQPA GI50 values for the 4 most sensitive SCLC lines were 11-30 nM and < 25% of untreated control growth was observed at 60 nM. Five lines had GI50 values of 30-60 nM but at 600 nM AZD1152-HQPA cell viability remained at 49-55% of untreated control growth. In the remaining 6-lines cell viability at 600 nM was 60-93% of control growth. Growth inhibition marginally correlated with MYC amplification (p=0.044) and was strongly correlated with MYC + MYCL1 + MYCN amplification (p=0.011). Apoptosis (20-28%) was induced by 30 nM AZD1152-HQPA at 24-48 hours in the most sensitive cell lines. Marked increases in DNA ploidy (8N) was observed after 24 hour exposure to AZD1152-HQPA (30nM) in the most sensitive cell lines and at 48 hours in the 5 intermediate lines and in 2 of the resistant lines. In vivo AZD1152 (i.p. 100 mg/kg/M-F for two weeks) induced tumor regression in nude mice implanted with a sensitive cell line. AZD1152 at 50mg/kg/day inhibited tumor growth; however these tumors began growing following treatment cessation. A resistant line was also implanted into nude mice and AZD1152 at 50 and 100 mg/kg/day caused tumor regression. Polyploidy was induced in this cell line at 48 hours post 30 nM AZD1152-HQPA. Anchorage independent growth in this resistant line was also completely inhibited by AZD1152-HQPA 25 nM. The doses of AZD1152-HQPA used in this study are within the range reported to be clinically achievable.

      Conclusion
      Conclusions: AZD1152-HQPA growth inhibition significantly correlated with MYC-family amplification in SCLC lines but some SCLC lines without MYC-family amplification were also inhibited. Additional biomarkers are needed to identify SCLC patients most likely to respond to AZD1152.

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    P3.05 - Poster Session 3 - Preclinical Models of Therapeutics/Imaging (ID 159)

    • Event: WCLC 2013
    • Type: Poster Session
    • Track: Biology
    • Presentations: 1
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      P3.05-023 - Treatment with the mTOR kinase inhibitor CC-223 overcomes resistance to the epidermal growth factor receptor tyrosine kinase inhibitor erlotinib in non-small cell lung cancer cells (ID 3429)

      09:30 - 09:30  |  Author(s): Z. Zhang

      • Abstract

      Background
      Activation of the mTOR pathway is a common down-stream mechanism of resistance to Epidermal Growth Factor receptor (EGFR) tyrosine kinase inhibitors. CC-223 (Celgene) is an mTOR kinase inhibitor under clinical development. We evaluated CC-223 in combination with erlotinib to overcome resistance to EGFR tyrosine kinase inhibition in non-small cell lung cancer (NSCLC) cell lines and xenografts in nude mice.

      Methods
      A panel of 18 NSCLC cell lines was used to evaluate the effect of erlotinib and CC-223 treatment on cell growth using an MTT assay. Intermediately (IC50 >1 and <10 mM) and highly (IC50 >10 mM) resistant cell lines to erlotinib were used in analyses of additive/synergistic effects for the combination treatment with CC-223.

      Results
      CC-223 demonstrated anti-proliferative activity in NSCLC cell lines with various degrees of sensitivity as reflected in different IC50 values, ranging from 0.15 to 12 mM. In combination with erlotinib, CC-223 showed synergistic anti-proliferative effects in NSCLC cells resistant to erlotinib with combination indices as low as 0.1-0.2. In vivo studies in mice xenografts demonstrated a strong synergistic effect of the combination treatment of erlotinib and CC-223 with a 90% decrease of tumor volume compared to untreated and 88% compared to erlotinib treated for A549 cells. IHC analyses of apoptosis and proliferation in the tumors are ongoing; mature data will be presented at the meeting.

      Conclusion
      The mTOR kinase inhibitor CC-223 demonstrated anti-proliferative activity in a panel of NSCLC cell lines. In NSCLC cells resistant to the EGFR tyrosine kinase inhibitor erlotinib, combining erlotinib with CC-223 demonstrated a synergistic anti-proliferative effect. In vivo studies of tumors in xenografted mice also demonstrated synergistic anti-tumor effects with the combination treatment even in erlotinib resistant tumors. The present data suggest that mTOR inhibition using the mTOR kinase inhibitor CC-223 may be a therapeutic strategy to overcome resistance to EGFR tyrosine kinase inhibitors in NSCLC.