Genetic editing of colonic organoids provides a molecularly distinct and orthotopic preclinical model of serrated carcinogenesis

Tamsin R.M. Lannagan, Young K. Lee, Tongtong Wang, Jatin Roper, Mark L. Bettington, Lochlan Fennell, Laura Vrbanac, Lisa Jonavicius, Roshini Somashekar, Krystyna Gieniec, Miao Yang, Jia Q. Ng, Nobumi Suzuki, Mari Ichinose, Josephine A. Wright, Hiroki Kobayashi, Tracey L. Putoczki, Yoku Hayakawa, Simon J. Leedham, Helen E. Abud & 10 others Ömer H. Yilmaz, Julie Marker, Sonja Klebe, Pratyaksha Wirapati, Siddhartha Mukherjee, Sabine Tejpar, Barbara A. Leggett, Vicki L.J. Whitehall, Daniel L. Worthley, Susan L. Woods

Research output: Contribution to journalArticle

4 Citations (Scopus)

Abstract

Objective Serrated colorectal cancer (CRC) accounts for approximately 25% of cases and includes tumours that are among the most treatment resistant and with worst outcomes. This CRC subtype is associated with activating mutations in the mitogen-Activated kinase pathway gene, BRAF, and epigenetic modifications termed the CpG Island Methylator Phenotype, leading to epigenetic silencing of key tumour suppressor genes. It is still not clear which (epi-)genetic changes are most important in neoplastic progression and we begin to address this knowledge gap herein. Design We use organoid culture combined with CRISPR/Cas9 genome engineering to sequentially introduce genetic alterations associated with serrated CRC and which regulate the stem cell niche, senescence and DNA mismatch repair. Results Targeted biallelic gene alterations were verified by DNA sequencing. Organoid growth in the absence of niche factors was assessed, as well as analysis of downstream molecular pathway activity. Orthotopic engraftment of complex organoid lines, but not Braf V600E alone, quickly generated adenocarcinoma in vivo with serrated features consistent with human disease. Loss of the essential DNA mismatch repair enzyme, Mlh1, led to microsatellite instability. Sphingolipid metabolism genes are differentially regulated in both our mouse models of serrated CRC and human CRC, with key members of this pathway having prognostic significance in the human setting. Conclusion We generate rapid, complex models of serrated CRC to determine the contribution of specific genetic alterations to carcinogenesis. Analysis of our models alongside patient data has led to the identification of a potential susceptibility for this tumour type.

LanguageEnglish
Pages684-692
Number of pages9
JournalGut
Volume68
Issue number4
DOIs
Publication statusPublished - 1 Apr 2019

Keywords

  • cancer genetics
  • colorectal cancer
  • gene mutation
  • methylation
  • oncogenes

ASJC Scopus subject areas

  • Gastroenterology

Cite this

Lannagan, T. R. M., Lee, Y. K., Wang, T., Roper, J., Bettington, M. L., Fennell, L., ... Woods, S. L. (2019). Genetic editing of colonic organoids provides a molecularly distinct and orthotopic preclinical model of serrated carcinogenesis. Gut, 68(4), 684-692. https://doi.org/10.1136/gutjnl-2017-315920
Lannagan, Tamsin R.M. ; Lee, Young K. ; Wang, Tongtong ; Roper, Jatin ; Bettington, Mark L. ; Fennell, Lochlan ; Vrbanac, Laura ; Jonavicius, Lisa ; Somashekar, Roshini ; Gieniec, Krystyna ; Yang, Miao ; Ng, Jia Q. ; Suzuki, Nobumi ; Ichinose, Mari ; Wright, Josephine A. ; Kobayashi, Hiroki ; Putoczki, Tracey L. ; Hayakawa, Yoku ; Leedham, Simon J. ; Abud, Helen E. ; Yilmaz, Ömer H. ; Marker, Julie ; Klebe, Sonja ; Wirapati, Pratyaksha ; Mukherjee, Siddhartha ; Tejpar, Sabine ; Leggett, Barbara A. ; Whitehall, Vicki L.J. ; Worthley, Daniel L. ; Woods, Susan L. / Genetic editing of colonic organoids provides a molecularly distinct and orthotopic preclinical model of serrated carcinogenesis. In: Gut. 2019 ; Vol. 68, No. 4. pp. 684-692.
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abstract = "Objective Serrated colorectal cancer (CRC) accounts for approximately 25{\%} of cases and includes tumours that are among the most treatment resistant and with worst outcomes. This CRC subtype is associated with activating mutations in the mitogen-Activated kinase pathway gene, BRAF, and epigenetic modifications termed the CpG Island Methylator Phenotype, leading to epigenetic silencing of key tumour suppressor genes. It is still not clear which (epi-)genetic changes are most important in neoplastic progression and we begin to address this knowledge gap herein. Design We use organoid culture combined with CRISPR/Cas9 genome engineering to sequentially introduce genetic alterations associated with serrated CRC and which regulate the stem cell niche, senescence and DNA mismatch repair. Results Targeted biallelic gene alterations were verified by DNA sequencing. Organoid growth in the absence of niche factors was assessed, as well as analysis of downstream molecular pathway activity. Orthotopic engraftment of complex organoid lines, but not Braf V600E alone, quickly generated adenocarcinoma in vivo with serrated features consistent with human disease. Loss of the essential DNA mismatch repair enzyme, Mlh1, led to microsatellite instability. Sphingolipid metabolism genes are differentially regulated in both our mouse models of serrated CRC and human CRC, with key members of this pathway having prognostic significance in the human setting. Conclusion We generate rapid, complex models of serrated CRC to determine the contribution of specific genetic alterations to carcinogenesis. Analysis of our models alongside patient data has led to the identification of a potential susceptibility for this tumour type.",
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Lannagan, TRM, Lee, YK, Wang, T, Roper, J, Bettington, ML, Fennell, L, Vrbanac, L, Jonavicius, L, Somashekar, R, Gieniec, K, Yang, M, Ng, JQ, Suzuki, N, Ichinose, M, Wright, JA, Kobayashi, H, Putoczki, TL, Hayakawa, Y, Leedham, SJ, Abud, HE, Yilmaz, ÖH, Marker, J, Klebe, S, Wirapati, P, Mukherjee, S, Tejpar, S, Leggett, BA, Whitehall, VLJ, Worthley, DL & Woods, SL 2019, 'Genetic editing of colonic organoids provides a molecularly distinct and orthotopic preclinical model of serrated carcinogenesis', Gut, vol. 68, no. 4, pp. 684-692. https://doi.org/10.1136/gutjnl-2017-315920

Genetic editing of colonic organoids provides a molecularly distinct and orthotopic preclinical model of serrated carcinogenesis. / Lannagan, Tamsin R.M.; Lee, Young K.; Wang, Tongtong; Roper, Jatin; Bettington, Mark L.; Fennell, Lochlan; Vrbanac, Laura; Jonavicius, Lisa; Somashekar, Roshini; Gieniec, Krystyna; Yang, Miao; Ng, Jia Q.; Suzuki, Nobumi; Ichinose, Mari; Wright, Josephine A.; Kobayashi, Hiroki; Putoczki, Tracey L.; Hayakawa, Yoku; Leedham, Simon J.; Abud, Helen E.; Yilmaz, Ömer H.; Marker, Julie; Klebe, Sonja; Wirapati, Pratyaksha; Mukherjee, Siddhartha; Tejpar, Sabine; Leggett, Barbara A.; Whitehall, Vicki L.J.; Worthley, Daniel L.; Woods, Susan L.

In: Gut, Vol. 68, No. 4, 01.04.2019, p. 684-692.

Research output: Contribution to journalArticle

TY - JOUR

T1 - Genetic editing of colonic organoids provides a molecularly distinct and orthotopic preclinical model of serrated carcinogenesis

AU - Lannagan, Tamsin R.M.

AU - Lee, Young K.

AU - Wang, Tongtong

AU - Roper, Jatin

AU - Bettington, Mark L.

AU - Fennell, Lochlan

AU - Vrbanac, Laura

AU - Jonavicius, Lisa

AU - Somashekar, Roshini

AU - Gieniec, Krystyna

AU - Yang, Miao

AU - Ng, Jia Q.

AU - Suzuki, Nobumi

AU - Ichinose, Mari

AU - Wright, Josephine A.

AU - Kobayashi, Hiroki

AU - Putoczki, Tracey L.

AU - Hayakawa, Yoku

AU - Leedham, Simon J.

AU - Abud, Helen E.

AU - Yilmaz, Ömer H.

AU - Marker, Julie

AU - Klebe, Sonja

AU - Wirapati, Pratyaksha

AU - Mukherjee, Siddhartha

AU - Tejpar, Sabine

AU - Leggett, Barbara A.

AU - Whitehall, Vicki L.J.

AU - Worthley, Daniel L.

AU - Woods, Susan L.

PY - 2019/4/1

Y1 - 2019/4/1

N2 - Objective Serrated colorectal cancer (CRC) accounts for approximately 25% of cases and includes tumours that are among the most treatment resistant and with worst outcomes. This CRC subtype is associated with activating mutations in the mitogen-Activated kinase pathway gene, BRAF, and epigenetic modifications termed the CpG Island Methylator Phenotype, leading to epigenetic silencing of key tumour suppressor genes. It is still not clear which (epi-)genetic changes are most important in neoplastic progression and we begin to address this knowledge gap herein. Design We use organoid culture combined with CRISPR/Cas9 genome engineering to sequentially introduce genetic alterations associated with serrated CRC and which regulate the stem cell niche, senescence and DNA mismatch repair. Results Targeted biallelic gene alterations were verified by DNA sequencing. Organoid growth in the absence of niche factors was assessed, as well as analysis of downstream molecular pathway activity. Orthotopic engraftment of complex organoid lines, but not Braf V600E alone, quickly generated adenocarcinoma in vivo with serrated features consistent with human disease. Loss of the essential DNA mismatch repair enzyme, Mlh1, led to microsatellite instability. Sphingolipid metabolism genes are differentially regulated in both our mouse models of serrated CRC and human CRC, with key members of this pathway having prognostic significance in the human setting. Conclusion We generate rapid, complex models of serrated CRC to determine the contribution of specific genetic alterations to carcinogenesis. Analysis of our models alongside patient data has led to the identification of a potential susceptibility for this tumour type.

AB - Objective Serrated colorectal cancer (CRC) accounts for approximately 25% of cases and includes tumours that are among the most treatment resistant and with worst outcomes. This CRC subtype is associated with activating mutations in the mitogen-Activated kinase pathway gene, BRAF, and epigenetic modifications termed the CpG Island Methylator Phenotype, leading to epigenetic silencing of key tumour suppressor genes. It is still not clear which (epi-)genetic changes are most important in neoplastic progression and we begin to address this knowledge gap herein. Design We use organoid culture combined with CRISPR/Cas9 genome engineering to sequentially introduce genetic alterations associated with serrated CRC and which regulate the stem cell niche, senescence and DNA mismatch repair. Results Targeted biallelic gene alterations were verified by DNA sequencing. Organoid growth in the absence of niche factors was assessed, as well as analysis of downstream molecular pathway activity. Orthotopic engraftment of complex organoid lines, but not Braf V600E alone, quickly generated adenocarcinoma in vivo with serrated features consistent with human disease. Loss of the essential DNA mismatch repair enzyme, Mlh1, led to microsatellite instability. Sphingolipid metabolism genes are differentially regulated in both our mouse models of serrated CRC and human CRC, with key members of this pathway having prognostic significance in the human setting. Conclusion We generate rapid, complex models of serrated CRC to determine the contribution of specific genetic alterations to carcinogenesis. Analysis of our models alongside patient data has led to the identification of a potential susceptibility for this tumour type.

KW - cancer genetics

KW - colorectal cancer

KW - gene mutation

KW - methylation

KW - oncogenes

UR - http://www.scopus.com/inward/record.url?scp=85049146061&partnerID=8YFLogxK

U2 - 10.1136/gutjnl-2017-315920

DO - 10.1136/gutjnl-2017-315920

M3 - Article

VL - 68

SP - 684

EP - 692

JO - Gut

T2 - Gut

JF - Gut

SN - 0017-5749

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ER -