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Tetsuya Mitsudomi



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    ES28 - Targeting KRAS (ID 245)

    • Event: WCLC 2020
    • Type: Educational Session
    • Track: Targeted Therapy - Clinically Focused
    • Presentations: 1
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      ES28.01 - Biology of KRAS Targeting Agents (ID 4118)

      15:30 - 16:30  |  Presenting Author(s): Tetsuya Mitsudomi

      • Abstract
      • Slides

      Abstract

      KRAS biology 1-3

      KRAS gene encodes for a 21kDa protein that toggles GDP-bound inactive conformation to and from GTP-bound active conformation. When RAS receives an upstream signal from receptor tyrosine kinases via guanine nucleotide-exchange factors (GEFs), GDP of inactive RAS is exchanged with GTP, resulting in an inactive form of RAS, which activates downstream pathways. GTP- bound RAS returns to GDP-bound RAS by its intrinsic GTPase activity. These processes are called as “RAS cycle.” GTPase activating proteins (RAS-GAPs, NF1) bind to active RAS and stimulate their GTPase activity by several magnitude orders. Mutations at codons 12, 13, and 61 of RAS disrupt GAP-mediated GTP hydrolysis. It is known that active RAS interacts with at least 20 effector proteins and stimulates downstream signaling cascades 4. Among them, RAF proteins activate the MAPK/ERK pathway resulting in cell proliferation. RalGDS activates small GTPases RalA and RalB that mediate cell transformation and cytoskeletal reorganization. PI3Ks activate the Akt family that plays a vital role in I cell survival, growth, and migration. The Association of RAS proteins with the membrane is essential for activating downstream signaling. This process includes farnesylation, proteolytic cleavage of the CAAX motif, carboxymethylation of the terminal Cys, and palmitoylation 1.

      RAS gene activation in lung cancer

      KRAS mutations usually occur in adenocarcinoma, especially with mucus production/goblet cell morphology5. They are more frequent in Caucasians (~30%) than East Asians (~10%)6 and are associated with smoking exposure7. KRAS mutation in lung cancer is characterized by the frequent a G to a T transversion in contrast to the frequent a G to an A transition in colorectal cancer8. There are at least six amino acid substitutions in KRAS mutation at codon 12. Mutant KRAS, in general, has weaker GTPase activity; however, G12C has near wild type GTPase activity despite its reduced GAP mediated hydrolysis 3. In contrast, KRAS G13D has elevated intrinsic nucleotide exchange activity3. Besides, KRAS subclassification according to co-mutation of LKB1 (the KL subgroup), TP53(KP), and CDKN2A/B with low TTF1 (KC) has been proposed, which reflect different biology, patterns of immune-system engagement, and therapeutic vulnerabilities 9. The prognostic impact of KRAS mutations in lung cancer is variably reported, but in general, it is thought to be a weak negative prognostic factor 10.

      How to target KRAS mutated lung cancer

      It appears that not all cancers with KRAS mutations are dependent on mutant KRAS. Upon treatment of shRNAs to deplete KRAS in lung cancer cell lines harboring KRAS mutations, cell lines with mesenchymal differentiation maintain viability without expressing KRAS11. This makes it challenging to develop a treatment strategy against KRAS mutated tumors.

      The early efforts to make RAS a druggable target include inhibition of membrane binding of RAS. This process is complicated involving several molecules such as farnesyl transferase (FT), geranylgeranyl transferase (GGT), ras-converting enzyme (RCE1), isoprenylcysteine carboxyl methyltransferase (ICMT) and also varies depending on different RAS molecules (HRAS, KRAS 4a, 4b (two KRAS isoforms), NRAS). With inhibition of FT, KRAS but not HRAS could be alternatively prenylated by GGT. Salirasib, farnesyl thiosalicylic acid, inhibits prenylated protein methyltransferase (PPMTase) with potent in vitro activity, including against KRAS; however, failed in a phase II trial12.

      Although RAF-MEK-ERK signaling is an essential downstream pathway of RAS, RAF or MEK inhibitors have been evaluated. For example, the phase 3 trial of MEK inhibitor selumetinib 13 did not show its benefit. This is partly because inhibition of MEK relieves negative feedback from ERK at multiple MAPK signaling levels, leading to re-activation of this pathway 14.

      There have been efforts to identify synthetic lethal interactions in cancer cells with KRAS mutation. In other words, it is to find which genes, when silenced by siRNA, kill cells harboring mutant RAS gene but not cells without this mutation. The list of genes with synthetic lethal activity against RAS mutated tumors are expandingand include THOC1, eNOS, Myc, Survivin, STK33, PLK1, SYK, RON, integrin b6, TBK1, NFkB, WT1, PKC delta, CDK4, JNK, ATR, GATA2, CDK4 15. Based on these experiments, abemaciclib, a CDK4/6 inhibitor, plus erlotinib was compared with erlotinib in patients with KRAS mutation in a phase III trial. Although PFS and ORR were improved, the primary endpoint of OS was not met 16

      Attempt to directly compete with GTP as in the case of receptor tyrosine kinase inhibition is difficult because of the very high affinity between RAS and GTP. Recently, KRASG12C inhibitors bind covalently to the mutant cysteine residue and occupy a pocket in the switch II region of GDP -bound KRAS. Early results of clinical trials are promising. Other approaches include inhibitors of SOS1:RAS interaction, inhibition of SHP2 (SH2 containing phosphatase 2) that activates RAS-MAPK pathway, immunotherapy targeting mutant KRAS.

      The RAS targeted therapy has been challenging because 1) there are many RAS-like G proteins in human, 2) not all RAS-mutated tumors are addicted to mutated RAS, 3) pathways involving RAS is extremely complicated and redundant, including feedback loops 4) Different RAS mutations have the different conformation and different biochemical consequences. In this talk, I would like to summarize the RAS activation basics in lung cancer and RAS-targeted drug development history.

      1. Simanshu DK, et al.: Cell 170:17-33, 2017

      2. Friedlaender A, et al.: Cancer Treat Rev 85:101978, 2020

      3. Moore AR, et al.: Nat Rev Drug Discov 19:533-552, 2020

      4. Erijman A, et al.: Mini Rev Med Chem 16:370-5, 2016

      5. Kobayashi T, et al.: Cancer 66:289-94, 1990

      6. Suda K, et al.: Cancer Metastasis Rev 29:49-60, 2010

      7. Slebos RJ, et al.: J Natl Cancer Inst 83:1024-7, 1991

      8. Bos JL: Cancer Res 49:4682-9, 1989

      9. Skoulidis F, et al.: Cancer Discov 5:860-77, 2015

      10. Mascaux C, et al.: British journal of cancer 92:131-9, 2005

      11. Singh A, et al.: Cancer Cell 15:489-500, 2009

      12. Riely GJ, et al.: J Thorac Oncol 6:1435-7, 2011

      13. Janne PA, et al.: JAMA 317:1844-1853, 2017

      14. Kitai H, et al.: Small GTPases 8:172-176, 2017

      15. Aguirre AJ, et al.: Cold Spring Harb Perspect Med 8, 2018

      16. Goldman JW, et al.: Front Oncol 10:578756, 2020

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    IS08 - Industry Symposium Sponsored by Janssen Oncology: What the Future Holds for Advanced NSCLC Patients with Resistance to EGFR TKIs (ID 285)

    • Event: WCLC 2020
    • Type: Industry Symposium
    • Track:
    • Presentations: 1
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      IS08.01 - Welcome and Introductions (ID 4341)

      13:00 - 14:00  |  Presenting Author(s): Tetsuya Mitsudomi

      • Abstract

      Abstract not provided

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    P76 - Targeted Therapy - Clinically Focused - EGFR (ID 253)

    • Event: WCLC 2020
    • Type: Posters
    • Track: Targeted Therapy - Clinically Focused
    • Presentations: 1
    • Moderators:
    • Coordinates: 1/28/2021, 00:00 - 00:00, ePoster Hall
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      P76.71 - RYK Confers Drug Tolerance to Osimertinib in Lung Cancer Cells with EGFR Mutations (ID 3355)

      00:00 - 00:00  |  Author(s): Tetsuya Mitsudomi

      • Abstract
      • Slides

      Introduction

      Emergence of acquired resistance is almost inevitable during EGFR-tyrosine kinase inhibitor therapy for non-small-cell lung cancer (NSCLC) harboring EGFR mutations. Drug tolerance, a reversible state of drug insensitivity in the early phases of tyrosine kinase inhibitor therapy, is considered to serve as the basis of recurrent disease. Therefore, it is important to elucidate the molecular mechanisms of drug tolerance.

      Methods

      Five EGFR-mutant NSCLC cell lines were used in this study. We established drug-tolerant cells (DTCs) via 72 h treatment with osimertinib (600 nM) or afatinib (60 nM). Acquisition of drug tolerance was evaluated by growth inhibitory assay, and the molecular mechanisms of drug tolerance were analyzed by phospho-RTK array.

      Results

      DTCs were successfully induced in PC9, HCC4006, and H1975 cells against osimertinib and in PC9 cells against afatinib. Next, we compared the phosphorylation levels of EGFR in HCC4006 cells after exposure to 600 nM osimertinib or 60 nM afatinib. 600 nM osimertinib inhibited the phosphorylation of EGFR in HCC4006 cells more than 60 nM afatinib. This was inconsistent with the experiments using PC9 cells (both 600 nM osimertinib and 60 nM afatinib induced drug tolerance in PC9 cells), showing that inhibition of EGFR phosphorylation was similar between 600 nM osimertinib and 60 nM afatinib.To investigate if the suppression level of EGFR phosphorylation was related to the acquisition of a drug-tolerant phenotype, we exposed HCC4006 cells to different concentrations of osimertinib or afatinib. Lower doses of osimertinib (66 nM and 200 nM) failed to induce a drug-tolerant state in HCC4006 cells, although the growth inhibition rate of 66 nM osimertinib for HCC4006 parental cells was identical to that of 600 nM osimertinib. However, higher doses of afatinib (180 nM and 540 nM) induced a drug-tolerant state in HCC4006 cells. We also observed that lower doses of osimertinib and afatinib failed to induce a drug-tolerant state in PC9 and H1975 cells. These results indicated that stronger inhibition of phosphorylated EGFR is necessary to induce DTCs. Next, in the analysis of HCC4006 DTCs against osimertinib, we observed increased receptor-like tyrosine kinase (RYK) expression, and siRNA-mediated RYK knockdown inhibited the proliferation of DTCs. This phenomenon was HCC4006 cell line-specific since we observed that PC9 parental cells and PC9-600 nM osim-DTCs were both resistant to RYK knockdown. In addition, cabozantinib, which reportedly inhibit RYK, monotherapy effectively inhibited the proliferation of HCC4006-600 nM osim-DTCs but not HCC4006 parental cells. However, cabozantinib was not active against PC9-600 nM osim-DTCs and PC9 parental cells.

      Conclusion

      Our study found that the inducibility of DTCs depended on the type of cell line and the drug concentration. It is possible that the optimal dose of EGFR-TKI in each patient may reduce the emergence of DTCs in clinical practice. We also found high expression of RYK was a molecular mechanism of the drug-tolerant state in HCC4006 cells against osimertinib. Further studies are necessary to fully understand the DTCs that are essential for the appropriate primary double-strike therapy for lung cancers with EGFR mutations.

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    P86 - Targeted Therapy - Clinically Focused - New Target (ID 263)

    • Event: WCLC 2020
    • Type: Posters
    • Track: Targeted Therapy - Clinically Focused
    • Presentations: 1
    • Moderators:
    • Coordinates: 1/28/2021, 00:00 - 00:00, ePoster Hall
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      P86.05 - In Vitro Validation Study for HER2 Mutations Identified in Secondary Analysis of the LUX-Lung 8 Randomized Clinical Trial (ID 3274)

      00:00 - 00:00  |  Author(s): Tetsuya Mitsudomi

      • Abstract
      • Slides

      Introduction

      LUX-Lung 8 demonstrated prolonged overall survival in patients with metastatic squamous cell carcinoma of the lung treated with afatinib compared to those treated with erlotinib. Its secondary analysis suggested that the patients with rare HER2 mutations contributed to the superiority of afatinib, since afatinib but not erlotinib has HER2 inhibitory activity (JAMA Oncol. 2018;4:1189-1197). However, transforming ability of all detected HER2 mutations but one has not been explored.

      Methods

      We have introduced ten HER2 mutations (Q57R, G152A, S250C, E265K, E395K, P489L, R683L, G815R, R929W and P1037L) into mouse pro-B cell line (Ba/F3). Since Ba/F3 requires interleukin 3 (IL-3) for its growth, IL-3 independent growth of Ba/F3 indicates that transduced mutation has transforming ability. For Ba/F3 cells which acquired IL-3 independent growth, we analyzed the efficacy of six EGFR or pan-ERBB tyrosine kinase inhibitors (TKIs) including afatinib, erlotinib, dacomitinib, neratinib, poziotinib, and osimertinib using growth inhibitory assay. The efficacy of six TKIs was compared using a sensitivity index as IC50 (50% inhibitory concentration) divided by the trough concentration at the recommended dose.

      Results

      figure 1.jpgSeven (Q57R, G152A, S250C, E265K, P489L, R683L and P1037L) out of ten Ba/F3 cells expressing HER2 mutations did not grow in the absence of IL-3, indicating that they were passenger or non-pathogenic mutations. On the other hand, three Ba/F3 cells expressing one of E395K, G815R or R929W acquired IL-3 independent growth, suggesting their transforming ability. The sensitivity indices for afatinib were five or more times higher than those of erlotinib in all three Ba/F3 lines (Figure 1). In the secondary analysis of the LUX-Lung 8 trial, patients with E395K and G815R mutations received afatinib, and survived without disease recurrence for 17 months and 6 months, respectively. The patient with R929W mutation received erlotinib, and survived without disease recurrence for only 2 months. Dacomitinib, neratinib, poziotinib, and osimertinib also effectively inhibited the growth of these three Ba/F3 lines.

      Conclusion

      HER2 E395K, G815R and R929W mutations had transforming ability, and 2nd/3rd generation TKIs, including afatinib, showed higher efficacy compared with erlotinib. These results may support the search of HER2 mutation in lung squamous cell carcinoma patients.

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    PL01 - Opening Plenary Session (Japanese, Mandarin, Spanish Translation Available) (ID 140)

    • Event: WCLC 2020
    • Type: Plenary
    • Track: N.A.
    • Presentations: 1
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      PL01.09 - Chair (ID 4396)

      18:00 - 20:00  |  Presenting Author(s): Tetsuya Mitsudomi

      • Abstract

      Abstract not provided

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    PS01 - Presidential Symposium (Japanese, Mandarin, Spanish Translation Available) (ID 143)

    • Event: WCLC 2020
    • Type: Plenary
    • Track: N.A.
    • Presentations: 1
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      PS01.14 - Chair (ID 4397)

      07:00 - 09:00  |  Presenting Author(s): Tetsuya Mitsudomi

      • Abstract

      Abstract not provided

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    PS02 - Presidential Symposium (Re-Broadcast) (Japanese, Mandarin, Spanish Translation Available) (ID 275)

    • Event: WCLC 2020
    • Type: Plenary
    • Track: N.A.
    • Presentations: 1
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      PS02.14 - Chair (ID 4398)

      18:00 - 20:00  |  Presenting Author(s): Tetsuya Mitsudomi

      • Abstract

      Abstract not provided