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M. Azab



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    O08 - Preclinical Therapeutic Models I (ID 92)

    • Event: WCLC 2013
    • Type: Oral Abstract Session
    • Track: Biology
    • Presentations: 1
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      O08.06 - Combining the HSP90 inhibitor, AT13387, with crizotinib improves response in an ALK-positive model of NSCLC. (ID 2163)

      17:00 - 17:10  |  Author(s): M. Azab

      • Abstract
      • Presentation
      • Slides

      Background
      Activation of anaplastic lymphoma kinase (ALK) via the EML4 translocation occurs in a proportion of non-small cell lung cancers (NSCLC). Whilst inhibitors of ALK such as crizotinib have been successful in the clinic, most patients ultimately relapse due to resistance via a number of different mechanisms. EML4-ALK and many critical components of signalling pathways involved in resistance are clients for the chaperone HSP90. This offers an alternative approach for targeting both ALK inhibitor-sensitive and -resistant disease through inhibition of HSP90 alone or in combination with an ALK inhibitor. AT13387 is a potent second-generation HSP90 inhibitor currently being clinically tested in a number of indications, including ALK-positive NSCLC as single-agent and in combination with the ALK inhibitor, crizotinib. Here we describe its activity in preclinical models of ALK-positive NSCLC and investigate its potential in combination with crizotinib.

      Methods
      The activity of AT13387 was investigated in vitro in the EML4-ALK translocated H2228 cell line. Protein levels were determined by western blotting. In vivo, AT13387 was evaluated in an H2228 tumor xenograft and an EML4-ALK translocated patient-derived xenograft model by measuring inhibition of tumor growth.

      Results
      AT13387 potently inhibited the proliferation of the crizotinib-sensitive EML4-ALK NSCLC cell line, H2228, in vitro with an IC~50~ value of 69 nM. The HSP90 client proteins, EML4-ALK and AKT, along with their phospho-forms, were depleted on treatment of these cells with AT13387. A simultaneous reduction in levels of phospho-ERK, phospho-AKT and phospho-S6 indicated that ALK signalling was inhibited, whilst induction of HSP70 confirmed HSP90 inhibition. In vivo, AT13387 demonstrated activity in ALK-dependent xenograft models, including an ALK-dependent patient-derived xenograft model. When mice bearing H2228 tumor xenografts were treated with AT13387 (70 mg/kg or 55 mg/kg ip once weekly), significant inhibition of tumor growth was observed. As expected, treatment with crizotinib (50 mg/kg po daily) caused partial tumor regression in this model (75% regression after 8 weeks of treatment). However, when AT13387 (55mg/kg weekly) was combined with the crizotinib treatment, a further enhancement of the inhibition of tumor growth over either of the monotherapies (88 % regression after 8 weeks) was observed, with 5 out of 7 tumors achieving complete regression, suggesting that the upfront addition of AT13387 to crizotinib treatment could lead to an improved response and potentially delay the emergence of resistance. In addition, this combination was well-tolerated.

      Conclusion
      AT13387 was shown to be effective in models of ALK-positive NSCLC as monotherapy or in combination with crizotinib, supporting the ongoing Phase II trial of AT13387 in ALK-positive NSCLC as single agent and in combination with crizotinib. These data suggest that treatment with an HSP90 inhibitor such as AT13387, alone or in combination with crizotinib, has therapeutic potential in ALK-positive NSCLC and that, furthermore, upfront combination of the two agents could extend the duration of response.

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    P1.05 - Poster Session 1 - Preclinical Models of Therapeutics/Imaging (ID 156)

    • Event: WCLC 2013
    • Type: Poster Session
    • Track: Biology
    • Presentations: 1
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      P1.05-012 - The HSP90 inhibitor, AT13387, displays single agent activity in erlotinib-sensitive and -resistant models of EGFR-activated NSCLC (ID 1791)

      09:30 - 09:30  |  Author(s): M. Azab

      • Abstract

      Background
      Epidermal growth factor receptor (EGFR) is activated in subsets of non-small cell lung cancer (NSCLC) by mutations such as L858R and exon19 deletions. EGFR-tyrosine kinase inhibitors such as gefitinib and erlotinib have been successfully used to treat tumors with these mutations, but responses tend to be limited by the development of resistance, often through further mutations in EGFR such as T790M. EGFR and its mutated forms are clients of HSP90 and so dependent on this chaperone for their stability. HSP90 inhibition is therefore an alternative mechanism for targeting EGFR-driven disease, which should be effective on EGFR inhibitor-sensitive or -resistant disease alike. AT13387 is a novel, potent, fragment-derived HSP90 inhibitor and is the subject of a number of Phase II clinical trials, including one in NSCLC.

      Methods
      The activity of AT13387 was investigated in vitro and in vivo in erlotinib-sensitive and -resistant EGFR-activated NSCLC cell lines and mouse xenograft models (see Table). The HCC827R cell line was generated by prolonged incubation of HCC827 cells with erlotinib. Cell proliferation was measured by Alamar blue assay. Protein levels were determined by western blotting.

      Results
      AT13387 was tested in a panel of EGFR-driven NSCLC cell lines and potently inhibited proliferation of both erlotinib-sensitive and -resistant cells including a cell line with acquired erlotinib resistance (HCC827R) (see Table).

      Inhibition of proliferation of EGFR-dependent NSCLC cell lines
      Cell line EGFR mutation status Erlotinib inhibition of proliferation IC50 (nM) AT13387 inhibition of proliferation IC50 (nM)
      HCC827 Exon19 Del 57 33
      NCI-H1650 Exon19 Del > 10 000 54
      NCI-H1975 L858R/T790M > 10 000 30
      H820 Exon19 Del/T790M > 10 000 70
      HCC827R N/D > 10 000 26
      Treatment of both erlotinib-sensitive and -resistant cell lines with AT13387 resulted in depletion of EGFR and its phospho-form, irrespective of its mutation status (L858R, T790M, Exon19 deletion). Other clients such as AKT were also depleted. A decrease in the levels of phospho-ERK and phospho-S6 indicated that EGFR signalling was also being inhibited in both erlotinib-sensitive and -resistant cells. In vivo, AT13387 significantly inhibited tumor growth in EGFR-driven tumor xenograft models (HCC827, NCI-H1975) when administered at 70 mg/kg ip once weekly. As expected, erlotinib dosed at 12.5 mg/kg once daily caused regression in HCC827 xenografts, whilst 75 mg/kg once daily had no effect on tumor growth in the resistant NCI-H1975 model. Levels of EGFR and phospho-EGFR were depleted for up to 72 hours in xenograft tumors treated with a single dose of 70 mg/kg AT13387, whilst a reduction in phospho-ERK and phospho-S6 again demonstrated an inhibition of signalling.

      Conclusion
      AT13387 was shown to be effective in erlotinib-sensitive and -resistant NSCLC models, depleting levels of EGFR regardless of its mutation status. These data suggest that AT13387 treatment may also be a potential approach for combating EGFR inhibitor resistance in the clinic.