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Yusuke Inoue

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    ES11 - Lung Cancer Plasticity and Drug Resistance (ID 14)

    • Event: WCLC 2019
    • Type: Educational Session
    • Track: Biology
    • Presentations: 1
    • Now Available
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      ES11.02 - SCLC Lineage Transformation in Lung Adenocarcinoma and Resistance to Targeted Therapies (Now Available) (ID 3212)

      15:15 - 16:45  |  Author(s): Yusuke Inoue

      • Abstract
      • Presentation
      • Slides


      Lung cancers are typically divided into two main histological types: small cell lung cancer (SCLC) and non-small cell lung cancer (NSCLC). NSCLC accounts for approximately 85% of all cases and lung adenocarcinoma (LUAD) is the most frequent subtype. Current treatments of LUAD aim to inhibit driver oncogene alterations and have shown unprecedented success. Among the oncogenic alterations in LUAD, EGFR kinase domain mutations are found in ~40-50% of patients in east-Asian countries and 20% of patients in western countries1. EGFR tyrosine kinase inhibitor (TKIs) are highly effective for tumors with EGFR mutations and resistance mechanisms to these compounds have been well documented: the most frequent being the acquisition of a secondary mutation in EGFR (T790M)2, followed by amplification of the hepatocyte growth factor receptor (MET) gene3 and mutations in BRAF and PIK3CA genes4,5. Histological transformation from LUAD to SCLC occurs in up to 15% of cases with acquired resistance to first and second generation EGFR TKIs5. Histological plasticity as a mechanism of resistance is becoming increasingly prominent as other resistant mechanisms can now be successfully targeted: MET-inhibitors are employed for MET-amplified tumours and 3rd generation EGFR TKIs are used to overcome resistance driven by the EGFR T790M muation6. Importantly, the 3rd generation EGFR TKI osimertinib was approved by the FDA in 2018 and thus, cases of treatment-induced SCLC transformation may increase in prominence as other mechanisms are targeted. Currently, conventional platinum doublet chemotherapy is the standard of care for patients with treatment-induced SCLC as well as de novo SCLC. Unfortunately, this treatment often produces an incomplete and non-durable response followed by inevitable relapse within months, leading to poor patient outcomes7. Thus, this mechanism of resistance will represent a major barrier towards the success of 3rd generation TKIs and new strategies to prevent this lineage shift or to treat SCLC transformed tumors are urgently needed.

      Despite the increasing clinical importance of LUAD to SCLC transformation, the biological pathways regulating this process are poorly understood. Since the first description in 20068, numerous studies have aimed to characterize the molecular changes that drive transformation in the context of drug resistance. Assessment of clinical samples has revealed that EGFR-mutant tumors universally lose EGFR protein expression upon SCLC transformation, despite still harboring EGFR mutations that confirms their clonal origin9. Furthermore, the mutation spectrum of these transformed cases often resemble de novo SCLC, containing inactivation of the tumor suppressors RB and p53 in nearly all cases9. This mirrors neuroendocrine transformation that occurs in prostate adenocarcinoma, where loss of RB/p53 are known to upregulate the reprogramming transcription factor SOX2, driving lineage plasticity and resistance upon anti-androgen therapy10. Furthermore, loss of RB and inactivation of p53 are required to reprogram a normal cell of epithelial lineage to a neuroendocrine lineage, and when combined with expression of myristoylated AKT1 and overexpression of MYC and BCL2, leads to the development of lethal SCLC in vivo11. Inactivation of p53 and RB also leads to the development of SCLC in transgenic mouse models, even when targeted in specifically to type-II airway epithelial cells, the putative cell of origin for EGFR-mutant LUAD12. Together, these studies highlight the essential role for these tumor suppressor genes in reprograming transcriptional profiles and chromatin accessibility in facilitating neuroendocrine lineage transformation.

      However, accumulating experimental evidence has demonstrated that while necessary, dual inactivation of RB and p53 is not sufficient to cause SCLC lineage transformation in EGFR-mutated LUAD, suggesting that additional factors are required9. MYC amplification and PIK3CA mutation have been proposed to potentially cooperate with RB/p53 loss to facilitate transformation13, and specific epigenetic regulators may also provide the appropriate context for lineage reprograming to occur. Despite this, no in vitro or in vivo models of SCLC transformation in EGFR TKI resistance have been developed, making it difficult to comprehensively explore the molecular events driving this lineage shift. Interestingly, there are clear differences between LUAD and SCLC regarding EGFR expression and gene alterations in MAPK pathway including EGFR/KRAS mutations: EGFR is usually not expressed14 and EGFR/KRAS mutations are extremely rare in SCLC15; in contrast, EGFR/KRAS play crucial roles in LUAD biology, including regulating differentiation in addition to prolferation16. To date, however, no clear explanation has been given for these differences. We have recently shown that activation of MAPK signaling in SCLC leads to suppression of the neuroendocrine phenotype - including downregulation of the transcription factors NEUROD1, INSM1, BRN2 and ASCL1 - and transformation to a NSCLC-like state17. Using this model system, we have begun to elucidate the key transcription factors and epigenetic changes that drive SCLC to NSCLC transformation in the hope that the same processes will also be involved in the clinically relevant scenario: SCLC transformation from EGFR mutant LUAD during TKI resistance. We suggest that only EGFR-mutant LUADs that do not reactivate MAPK signaling through secondary EGFR mutations or alterations in parallel kinase pathways (ie. MET) during development of TKI resistance will be able to undergo SCLC lineage transformation, and that RB/p53 loss and epigenetic plasticity provide the permissive context for which this transformation can occur. Greater understanding of lineage transformation in LUAD will provide important insights in terms of managing outcomes of patients undergoing targeted therapy and offer new avenues towards treatment of TKI resistant tumors.


      1. Dearden S et al. Ann Oncol 2013;24:2371-6.

      2. Kobayashi S et al. NEJM 2005;352:786-92.

      3. Bean J et al. PNAS 2007;104:20932-7.

      4. Ohashi K et al. PNAS 2012;109:E2127-33.

      5. Sequist LV et al. Sci Transl Med 2011;3:75ra26.

      6. Mok TS et al. NEJM 2017;376:629-40.

      7. Roca E et al. Cancer Treat Rev 2017;59:117-22.

      8. Zakowski MF et al. NEJM 2006;355:213-5.

      9. Niederst MJ et al. Nat Commun 2015;6:6377.

      10. Mu P et al. Science 2017;355: 84-8.

      11. Park et al. Science. 2018;362:91-95.

      12. Sutherland KD et al. Cancer Cell 2011;19:754-64

      13. Lee JK et al. J Clin Oncol. 2017;35:3065-3074.

      14. Gamou S et al. Cancer Res 1987;47:2668-73.

      15. Cristea S et al. J Thorac Oncol 2016;11:1233-41.

      16. Byers LA et al. Cancer Discov 2012;2:798-811.

      17. Y. Inoue and W. Lockwood. J Thorac Oncol 2018;13:S433–S434.

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    P2.03 - Biology (ID 162)

    • Event: WCLC 2019
    • Type: Poster Viewing in the Exhibit Hall
    • Track: Biology
    • Presentations: 1
    • Now Available
    • Moderators:
    • Coordinates: 9/09/2019, 10:15 - 18:15, Exhibit Hall
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      P2.03-26 - Elucidating Mechanisms of Resistance to Targeted Therapies in Mutant EGFR or KRAS Driven Lung Adenocarcinoma Harboring Dual Loss of p53 and RB1 (Now Available) (ID 2004)

      10:15 - 18:15  |  Presenting Author(s): Yusuke Inoue

      • Abstract
      • Slides


      Inactivation of the two canonical tumor suppressors, p53 and RB1, is a genetic hallmark of small-cell lung cancer (SCLC). In contrast, lung adenocarcinomas (LUADs) preferentially harbor alterations in the p16 pathway over RB1. Nonetheless, despite being rare, concurrent loss of p53 and RB1 occurs in a subset of LUADs and this is hypothesized to be necessary for the histological transformation of LUAD to SCLC, observed during treatment with tyrosine kinase inhibitors (TKIs). However, whether the dual loss of p53 and RB1 is sufficient for this histological transformation remains unknown. Furthermore, loss of RB1 in LUADs with EGFR mutations is associated with poor response to TKIs in the absence of SCLC transformation. Here, we aimed to explore how loss of p53/RB1 affects the biology of p16 pathway-altered LUAD, particularly in the context of acquired resistance mechanisms to targeted therapies.


      Four TP53-mutated LUAD cell lines were used: two EGFR mutation-positive (PC9 and H1975) and two KRAS mutation-positive (H1792 and H358). All these cell lines possess p16 pathway alterations: p16 (CDKN2A) mutations in PC9 and H1975, CDK4 amplification in H1792, and silenced p16 in H358. Inactivation of RB1 was carried out using CRISPR-Cas9 and RB1 knockout monoclonal cells were established. Cell proliferative and clonogenic abilities were assessed. In addition, osimertinib-resistant (PC9 and H1975) and trametinib-resistant (H1792 and H358) cells were generated (initial high dose and/or stepwise dose escalation methods). Acquired resistance mechanisms were evaluated by MSK-IMPACT profiling.


      Two RB1 knockout clones were established for each cell line. No advantageous effects were observed for proliferative and clonogenic abilities after RB1 knockout. Although loss of p53 and RB1 has been reported to result in lineage shift in prostate cancer through the upregulation of SOX2, deregulation of SOX2 expression was not observed upon RB1 knockout in the TP53-mutant LUAD cells. In addition, although loss of RB1 caused a modest reduction in osimertinib and trametinib sensitivities of H1975 and H358 cells, respectively, no effect was observed in PC9 and H1792 cells. After becoming resistant to osimertinib or trametinib, transformation to SCLC was not observed. Individual resistance mechanisms are currently being assessed by MSK-IMPACT.


      Dual loss of p53 and RB1 caused no advantageous effects in TP53-mutated and p16 pathway-altered LUAD cells and did not initiate transformation to SCLC as a resistance mechanism to targeted therapies. TP53/RB1 deficiency-related acquired resistance mechanisms to osimertinib or trametinib will be further explored and presented.

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