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Vijaya G Tirunagaru



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    P1.14 - Targeted Therapy (ID 182)

    • Event: WCLC 2019
    • Type: Poster Viewing in the Exhibit Hall
    • Track: Targeted Therapy
    • Presentations: 1
    • Moderators:
    • Coordinates: 9/08/2019, 09:45 - 18:00, Exhibit Hall
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      P1.14-20 - Tarloxotinib as a Novel Therapeutic Strategy for Oncogenic Alterations Across the ErbB Family of Receptors (ID 2607)

      09:45 - 18:00  |  Author(s): Vijaya G Tirunagaru

      • Abstract

      Background

      The ErbB family of receptors tyrosine kinases (EGFR, HER2, HER3, and HER4) have been implicated in multiple different tumor types. The implementation of comprehensive next generation sequencing has allowed the identification of diverse gene alterations that function as oncogene drivers in these receptors. Some of the non-common gene alterations identified are resistant to marketed EGFR/HER2 inhibitors. Tarloxotinib is a prodrug that generates a potent and irreversible pan-HER inhibitor (tarloxotinib-E) under hypoxic conditions associated with tumors. In this study we evaluated the effect of tarloxotinib on several types of oncogenic mutations and fusions that involve the ErbB family of receptors.

      Method

      cDNAs encoding EGFR kinase domain duplications (EGFR-EGFR), EGFR-RAD51 gene fusion, EGFR-ERBB4, ERBB2-GRB7 and EZR-ERBB4 gene fusion were expressed in Ba/F3 cells. Using spheroid assays we evaluated the proliferation of A172 glioblastoma cell line treated with tarloxotinib, tarloxotinib-E or 1st, 2ndor 3rdgeneration EGFR/HER2 TKIs. We analyze the on target and signaling effects elicited by tarloxotinib-E via immunoblots. Using a nude mice xenograft model of the human derived cell line CUTO17 wi the EGFR exon 20 insertion p.N771_H773dupNPH, we evaluated tumor, tissue and blood drug levels by mass spectrometry and the effect of tarloxotinib on tumor growth.

      Result

      Our results demonstrate that tarloxotinib-E inhibits phosphorylation of EGFR with a kinase duplication and inhibits proliferation in a spheroid invasion assay in A172 cells. In the CUTO17 EGFR exon 20 model, treatment with tarloxotinib inhibited tumor growth. Intratumor levels of tarloxotinib-E were ~20 times higher than skin and ~50 times higher than plasma demonstrating selective tumor conversion of tarloxotinib. Cell growth inhibition (EC50) of novel HER family fusions (EGFR-EGFR, EGFR-RAD51, EGFR-ERBB4, ERRB2-GRB7 and EZR-ERBB4) will be presented.

      Conclusion

      Tarloxotinib is a potent irreversible inhibitor in vitro for cells that harbor oncogenic alterations across the ERBB gene family, including EGFR kinase domain duplications, ErbB fusions and exon-20 insertions. Tarloxotinib is selectively activated in hypoxic tumor regions demonstrating a novel mechanism to generate a therapeutic window and avoid on-target EGFR-related toxicities.

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    P2.03 - Biology (ID 162)

    • Event: WCLC 2019
    • Type: Poster Viewing in the Exhibit Hall
    • Track: Biology
    • Presentations: 1
    • Moderators:
    • Coordinates: 9/09/2019, 10:15 - 18:15, Exhibit Hall
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      P2.03-20 - Potent in Vitro Activity of Tarloxotinib for HER2 Exon 20 Mutations in Lung Cancer and Mechanism of Acquired Resistance (ID 2093)

      10:15 - 18:15  |  Author(s): Vijaya G Tirunagaru

      • Abstract

      Background

      Oncogenic HER2 (ERBB2) mutations are present in 2-3% of lung adenocarcinoma. No targeted therapy is currently approved for HER2-mutated lung cancers, and clinical trials using novel irreversible pan-HER inhibitors are underway. However, all of these irreversible pan-HER inhibitors are also active against wild-type (WT) EGFR, resulting in dose limiting toxicities. Tarloxotinib is a novel clinical-stage prodrug that releases a potent, irreversible pan-HER inhibitor (tarloxotinib-E) selectively in pathologically hypoxic regions of tumors. In this study, we evaluated tarloxotinib-E activity against various HER2 exon20 insertion mutations and explored the resistance mechanisms to tarloxotinib-E.

      Method

      We introduced WT HER2 or HER2 activating mutations, including A775_G776insYVMA, G776delinsVC, and P780_Y781insGSP into Ba/F3 cells by retroviral transfection. Growth inhibitory assays were performed in these Ba/F3 cells and in H1781 cells (G776delinsVC). Tarloxotinib-E resistant clones were established by exposing these Ba/F3 cells to 200nM of tarloxotinib-E after treatment with N-ethyl-N-nitrosourea. Acquired resistant cells to tarloxotinib-E were also developed from H1781 cells via chronic exposure to increasing concentrations of tarloxotinib-E. HER2 secondary mutations were detected by direct sequencing.

      Result

      Tarloxotinib-E displayed potent activity against WT and mutant HER2 Ba/F3 cells and H1781 cells. Furthermore, the IC50 of tarloxotinib (prodrug) for wild-type HER2 was > 100 times higher than that of tarloxotinib-E (active drug). So far, we established 12 tarloxotinib-E resistant clones from Ba/F3 models (6 with A775_G776insYVMA and 6 with G776delinsVC), all of which harbored C805S secondary mutation (corresponding to C797S of the EGFR).tarlo ic50.jpg

      Conclusion

      Tarloxotinib-E exhibited potent activity for all HER2 exon 20 mutations. We identified secondary HER2 C805S mutation as a common mechanism of acquired resistance, consistent with the covalent binding mode of tarloxotinib via this residue. Additional resistant clones are currently being evaluated.

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    P2.14 - Targeted Therapy (ID 183)

    • Event: WCLC 2019
    • Type: Poster Viewing in the Exhibit Hall
    • Track: Targeted Therapy
    • Presentations: 1
    • Moderators:
    • Coordinates: 9/09/2019, 10:15 - 18:15, Exhibit Hall
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      P2.14-16 - T790M or C797S Confers Acquired Resistance to Tarloxotinib and Poziotinib in EGFR Exon 20 Insertion-Driven Lung Cancer Models in Vitro (ID 1694)

      10:15 - 18:15  |  Author(s): Vijaya G Tirunagaru

      • Abstract

      Background

      Lung cancers with EGFR exon 20 insertion mutations are refractory to current available tyrosine kinase inhibitors. Recent evidence suggests the therapeutic potential of the novel hypoxia-activated prodrug, tarloxotinib, a potent pan-HER inhibitor, or the repurposed 3rd generation inhibitor, poziotinib in these tumors. However, it is assumed that acquired resistance to these agents will be inevitable. In this study, we explored secondary mutations that may confer resistance to these agents using Ba/F3 models.

      Method

      Ba/F3 cells with various EGFR exon 20 mutations (A763insFQEA, V769insASV, D770insSVD, and H773insNPH) were generated by retroviral transfection. TKI resistant clones were established by exposure of parental cells to either tarloxotinib-E (active form of tarloxotinib) or poziotinib after treatment with N-ethyl-N-nitrosourea (ENU) mutagenesis agent. EGFR secondary mutations were detected by direct sequencing.

      Result

      ENU mutagenesis resulted in 62 tarloxotinib-E-resistant clones and 56 poziotinib-resistant clones using 200 nM of either drug. Ba/F3 cells with A763insFQEA were highly sensitive to these agents and only 1 tarloxotinib-resistant clone (C797S) was obtained. In Ba/F3 cells with V769insASV, all 14 tarloxotinib-resistant clones and 15 poziotinib-resistant clones developed T790M mutation. In Ba/F3 cells with D770insSVD cells, all 24 tarloxotinib-resistant clones and 20 poziotinib-resistant clones developed C797S mutation. In Ba/F3 cells with H773insNPH cells, 22 of 23 tarloxotinib-resistant clones and 21 of 22 poziotinib-resistant clones developed T790M mutation, while the rest of the clones harbored C797S mutation.

      Conclusion

      These results suggested that either T790M or C797S could cause acquired resistance to tarloxotinib and poziotinib in lung cancer models with EGFR exon 20 insertion mutations. Interestingly, the type of resistance mutation is likely to be dependent on the context of the original EGFR exon 20 insertion mutation.