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  MS 12 - NSCLC Stems Cells: Are They a Real Target? (ID 30)

  • Event: WCLC 2015
  • Type: Mini Symposium
  • Track: Treatment of Advanced Diseases - NSCLC
  • Presentations: 1
  • Moderators:C. Dive, K. Nakagawa
  • Coordinates: 9/08/2015, 14:15 - 15:45, Mile High Ballroom 2c-3c

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    MS12.03 - Where to Go from Here? (ID 1902)

    15:00 - 15:20  |  Author(s): I. Chaib
    • Abstract
    • Slides

    Abstract:
    Lung cancer is a dismal disease, however, anticipated selective responses are observed in a subgroup of non-small cell lung cancer (NSCLC) patients where the disease is driven by epidermal growth factor receptor (EGFR) mutations. EGFR mutations occur in 15 – 40% of lung adenocarcinomas, according to gender, smoking history and geographical region. Two types of EGFR mutations account for 90% of all lung adenocarcinoma-associated EGFR mutations and are related to sensitivity to treatment with oral tyrosine kinase inhibitors (TKIs), such as gefitinib, afatinib or AZD9291: (i) small in-frame deletions in exon 19 that lead to elimination of an LREA motif in the protein (DEL) and (ii) a point mutation in exon 21 that substitutes an arginine for a leucine at position 858 in the protein (L858R). Lung cancer patients bearing EGFR mutations show radiographic responses to TKIs in 60 – 70% of cases. Although the majority of patients achieve a significant therapeutic benefit, almost all invariably progress in less than 1 year. Therefore there is an unmet medical need for novel therapies in order to avoid resistance to treatment. We have employed a wide array of approaches (MTT, western blot analysis, PCR, Aldefluor assay and mouse models) to demonstrate that the combination of gefitinib, afatinib or AZD9291 with compounds targeting signal transducer and activator of transcription 3 (STAT3) can suppress the mechanisms of early adaptive resistance. STAT3 is a member of a family of proteins responsible for transmission of peptide hormone signals from the extracellular surface of the cells to the nucleus. STAT3 is a master regulator of several key hallmarks and enablers of cancer cells, including cell proliferation, resistance to apoptosis, metastasis, immune evasion, tumor angiogenesis, epithelial-mesenchymal transition, response to DNA damage and the Warburg effect. In addition STAT3 promotes an increase in the cell renewal of tumor-initiating cells or cancer stem cell subpopulation, mainly aldehyde dehydrogenase (ALDH). EGFR mutations cause receptor oligomerization and activation of intrinsic or receptor-associated tyrosine kinases, respectively. These activated kinases phosphorylate receptor tyrosine residues creating docking sites for recruitment of cytoplasmic STAT3. STAT3 docks to receptor phosphotyrosyl (pY) peptide sites through its Src-homology (SH2) domain which leads to its phosphorylation on Y705 followed by STAT3 tail-to-tail homodimerization (SH2 domain of each monomer binds to the pY peptide domain of each partner). STAT3 homodimers accommodate in the nucleus, where they bind to specific STAT3 response elements in the promotor of target genes and regulate their transcription. EGFR mutations and tyrosine kinase-associated receptor interleukin-6 (IL-6) lead to the activation of STAT3 that is not obliterated by EGFR TKIs. Even more, 2 hours after starting gefitinib treatment there is an increase in STAT3 activation in EGFR mutant cell lines (P. Ma, Cancer Research, 2011). Moreover, following erlotinib treatment there is an enrichment of ALDH+ stem-like cells through EGFR-dependent activation of Notch3. We have tested several small molecules that target STAT3. The combination inhibits cell viability in several human EGFR mutant cells and blocks STAT3 activation. However, neither the combination of EGFR TKIs with TPCA1 (repurposed as a STAT3 inhibitor), nor the combination of gefitinib with AZD0530 (a Src inhibitor) prevent the increment in the ALDH + cancer stem cell subpopulation. Therefore, we are exploring more in depth the crosstalk between EGFR and IL-6. As a whole, human EGFR mutant cell lines have increased levels of IL-6 which leads to STAT3 hyper-activation. Nevertheless, recent evidence indicates that IL-6-Src can induce YAP activation and NOTCH signaling. The downstream effectors of YAP and NOTCH ligands CTGF and HES1, respectively, are being examined in clinical tumor samples. We have examined the combination of Src, YAP and NOTCH inhibitors in addition to the use of STAT3 inhibitors. The triple combination of gefitinib plus TPCA1 plus AZD0530 had great synergism with a very low combination index and also eliminated the ALDH+ population (Figure). Furthermore, the overexpression of ALDH1A1 was decreased with the triple combination, however with only gefitinib plus TPCA1 or gefitinib plus AZD0530, ALDH1A1 mRNA was substantially increased in comparison with gefitinib alone (Figure). The western blot for the triple combination shows the inhibition of STAT3 Y705 phosphorylation as well as the phosphorylation of YAP (Ser397) and also from BMI1. We plan to confirm some of the data in clinical tumor samples to understand the contribution of IL6 and well established effectors-the SHP2-ERK, PI(3)K-Akt-mTORC1 and JAK-STAT3 modules and the interaction with YAP. Figure 1
    
    
    
    

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