Identification of novel determinants of primary and acquired resistance to EGFR-targeted therapies in colorectal cancer

Abstract

Globally, colorectal cancer (CRC) is the third most commonly diagnosed cancer, and the second leading cause of cancer related mortality – equating to approximately 16,000 deaths occurring within the UK between 2015 and 2017. While the mainstay of CRC treatment with regard to chemotherapeutic intervention is infusional 5-FU with folinic acid, this combination has been shown to elicit a response rate of only 10- 15% in stage IV CRC patients. The addition of oxaliplatin or irinotecan to 5-FU and folinic acid (FOLFOX and FOLFIRI respectively) was determined to increase this response rate and overall survival to 40-50% and 15-19 months respectively. These combination chemotherapeutics subsequently became the standard of care (SOC) treatment for stage IV CRC patients in the palliative and neo-adjuvant settings. In an effort to further increase efficacy and overall survival for these patients, targeted therapies have been developed which are directed at specific pathways implicated in the proliferation of cancer cells and propogation of the tumour. One major caveat of the use of these targeted therapies is the existence of resistance, rendering these treatments ineffective. Resistance towards targeted therapies can exist innately within the tumour or can develop over a period of time following prolonged exposure to the targeted therapy. The EGFR-targeted therapy cetuximab is a chimeric IgG1 monoclonal antibody which binds to the ligand binding domain of the receptor, thereby preventing activation of survival signalling via the MAPK pathway mediated by phosphorylation of EGFR. Cetuximab has been approved for use within stage IV CRC patients that do not exhibit a RAS or BRAF mutation. Despite this, it has been shown that while RAS/BRAFWT patients initially respond to cetuximab treatment, resistance to the EGFR mAb can occur ~3-18 months following initial challenge. With regard to both innate and acquired resistance towards the cetuximab, the most common driver of resistance is mutation of the KRAS gene, exhibited within ~48% CRC patients, which mediates constitutive activation of the MAPK survival pathway in a manner independent from EGFR-phosphorylation. Similarly, mutations in BRAF, PIK3CA and Although a number of resistance mechanisms to cetuximab treatment have been elucidated (in the context of both acute and acquired resistance), there still remains ~20% of patients that do not exhibit any of these alterations but remain unresponsive to the mAb. There is therefore a clear need for investigation into novel drivers of cetuximab resistance in CRC to identify effective treatment strategies for these patients.

We initially established that while the LIM1215 CRC cell line model did not exhibit any mutations in RAS, BRAF, PIK3CA or PTEN, treatment with cetuximab did not mediate an apoptotic effect, indicating this cell line was innately resistant towards the mAb. We subsequently identified that while there was abolition of EGFR phosphorylation within LIM1215 cells treated with cetuximab, there remained downstream activation of the MAPK pathway, as indicated by sustained ERK1/2 phosphorylation. We also determined that LIM1215 cells exhibited increased TGFα shedding following cetuximab treatment while another CRC cell line model that exhibited sensitivity towards the mAb did not. As we previously established that ERK1/2 phosphorylation can mediate activation of the metalloproteinase ADAM17, which has also been shown to mediated shedding of TGFα, we investigated if ADAM17 played a role in acute cetuximab resistance in this context. As such, we demonstrated that inhibition of ADAM17 either through siRNA silencing or with the use of an inhibitor (IK682) can increase sensitivity towards cetuximab within the LIM1215 CRC cell line. Furthermore, a phospho-RTK array performed on the conditioned media of LIM1215 cells in the presence or absence of cetuximab, also allowed us to establish a potential role for the HER3 RTK in cetuximab resistance within this cell line. This role of HER3 was further confirmed when siRNA silencing of the receptor was determined to abrogate the reactivation of ERK1/2 phosphorylation mediated by cetuximab treatment. We therefore concluded that both HER3 and ADAM17 are implicated in mediating innate cetuximab resistance within the LIM1215 CRC cell line, although further investigation is required to further elucidate the role of each of these drivers.

Within chapter 4, we sought to establish a novel therapeutic strategy to treat instances of both innate and aquired resistance to cetuximab. Due to the increased proliferation and prolonged survival of cetuximab resistant cells, we therefore decided to focus on a potential role of the unfolded protein response (UPR). We initially determined that CRC cell lines with an acquired KRAS alteration induced by prolonged cetuximab exposure, exhibited increased basal activation of the UPR compared to their KRASWT counterparts – in a similar manner to what we had previously determined in cell line models presenting with BRAF mutations. Furthermore, we established that CRC cell lines presenting with KRAS/BRAF alterations displayed higher levels of sensitivity to the imipriodone ONC201 in comparison to KRAS/BRAFWT cell line models. We subsequently demonstrated that combination of ONC201 and taxanes mediated an synergistic apoptotic effect, which was initially demonstrated in BRAFMT cell lines. This treatment strategy was also shown to display efficacy within LIM1215 CRC cells with an acquired KRAS mutation. This chapter therefore presents strong evidence that CRC cell lines with KRAS or BRAF mutations display chronic basal activation of the UPR. Treatment of these cells with acute activators of ER stress eg. the DRD2 antagonist ONC201, in combination with taxanes can be utilised as a novel treatment strategy. Within this chapter we also demonstrated a potential prognostic role for GRP78 expression within a cohort of 156 stage II/III CRC patients. We identified that KRASMT patients with high GRP78 expression had a significantly lower 5yr survival rates compared to KRASMT patients exhibiting low levels of GRP78 expression, however this result will require verification using a larger patient cohort.

Date of Award	Dec 2020
Original language	English
Awarding Institution	Queen's University Belfast
Sponsors	Northern Ireland Department for the Economy
Supervisor	Sandra Van Schaeybroeck (Supervisor), Liam Murray (Supervisor) & Ian Mills (Supervisor)