Exploiting genomic vulnerabilities in lung cancer for therapeutic targetting

Onderzoeksoutput: PhD Thesis

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Samenvatting

Non-small cell lung cancer (NSCLC) is a disease with high incidence and is
associated with high disease related mortality. A substantial subset of these lung cancers is driven by oncogenic mutations in various genes.
The first research chapter of this thesis focuses on the activating mutations
in the epidermal growth factor receptor (EGFR), which induce sensitivity to
EGFR tyrosine kinase inhibitors. Despite impressive clinical responses,
patients ultimately relapse as a reservoir of drug-tolerant cells persist,
which eventually leads to acquired resistance mechanisms. We performed a high-throughput siRNA screen to identify proteins that abrogate the response of EGFR-mutant NSCLC to EGFR-targeted therapy. The deubiquitinase (DUB) USP13 was a top hit resulting from this screen.
Targeting USP13 increases the sensitivity to EGFR inhibition with small
molecules in vitro and in vivo. USP13 selectively stabilizes mutant EGFR in
a peptidase-independent manner by counteracting the action of members
of the Cbl family of E3 ubiquitin ligases. We conclude that USP13 is a strong
mutant EGFR-specific co-target that could improve the treatment efficacy
of EGFR-targeted therapies.
In the second research chapter, we investigated the sensitivity of non-V600
BRAF driven NSCLC to type II RAF inhibitors together with MEK inhibitors.
We compared the effects of Dabrafenib and a type II RAF inhibitor, AZ628
on both BRAF class II (high kinase) and class III (kinase impaired) mutations
in non-V600 BRAF mutant expressing HEK293T cells and in a relevant BRAF
mutant NSCLC cell line (H1666). Importantly, we show that AZ628 did not
induce paradoxical ERK activation in CRAF (co-)expressing cells, in contrast
to dabrafenib. Overall, AZ628 inhibited ERK more effectively than
dabrafenib in both models (HEK293T and H1666). In combination with
trametinib, AZ628 showed better MEK-inhibitory, pro-apoptotic effects
and resulted in greater inhibition of cell growth compared to dabrafenib.
These results indicate that type II RAF inhibitors (such as AZ628) have
greater potential than dabrafenib, both as a single agent and combined
with trametinib, for the treatment of non-V600 BRAF mutant lung cancer.
In the last research chapter we investigated the impact of two CRAF
mutations discovered in two out of 41 NSCLC patients, namely CRAF P261A and CRAF P207S previously unreported in cancer. We show that CRAF
P261A is activated in a dimer-dependent manner, lacking S259 negative
regulatory phosphorylation. The CRAF P207S does not result in a gain-of-
function. Stable expression of the CRAF P261A in MEF and BEAS-2B cell
lines induced anchorage-independent growth. Additionally, we show that type II RAF inhibitors suppress ERK pathway activation by CRAF P261A in BEAS2B. Additionally, the combination with trametinib and type II RAF inhibitors led to a more potent ERK pathway inhibition and growth arrest in CRAF P261A expressing BEAS2B cells. Our findings suggest that the CRAF P261A mutation can provide oncogenic properties to cells and that such cells are sensitive to combined MEK and type II RAF inhibitors.
In conclusion, this PhD work contributes to the understanding and targeting of functional insensitivity to existing EGFR targeted therapies, further established safe and effective actionability of non-V600 BRAF mutations and identified a novel targetable driver CRAF in NSCLC which is also actionable.
Originele taal-2English
KwalificatieDoctor in Medical Sciences
Toekennende instantie
  • Vrije Universiteit Brussel
Begeleider(s)/adviseur
  • De Grève, Jacques, Promotor
  • Rooman, Ilse, Promotor
  • Gutierrez Gonzalez, Gustavo, Co-Promotor
  • Teugels, Erik, Co-Promotor
Datum van toekenning3 mei 2022
StatusPublished - 2022

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