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Page 282 Cancer Drug Resist 2018;1:266-302 I http://dx.doi.org/10.20517/cdr.2018.18

(PDAC) and drive tumorigenesis and disease progression. The PI3K pathway is activated downstream of
oncogenic KRAS signalling and presents an attractive therapeutic target for the treatment of this lethal
disease. However, when tested in PDAC clinical trials, PI3K pathway inhibitors have thus far derived no
significant benefit. We hypothesise that an adaptive response mediates resistance to PI3K inhibition in
PDAC, targeting of which may present a novel therapeutic strategy. We found that resistance to the PI3K
alpha inhibitor, BYL719, is associated with sustained PI3K-independent activation of mTORC1 in a panel of
PDAC cell lines. We demonstrate that inhibition of mTOR, in combination with PI3K inhibition, is neces-
sary to fully supress PI3K signalling and inhibit proliferation of PDAC cells. Through genome-scale and
focussed, mini-pool CRISPR screens, we nominated key genes in the mTOR and RTK signalling networks
responsible for mediating resistance and show that siRNA mediated knockdown of these genes is sufficient
to sensitise to PI3K alpha inhibition in multiple cell lines. Notably, over-expression of multiple genes in-
volved in determining PI3K inhibitor sensitivity is associated with significantly decreased patient survival.
Targeting of multiple nodes in the PI3K signalling network is therefore essential to overcoming resistance
to single agent PI3K alpha inhibition. In 3D stromal co-culture models, inhibition of either mTOR, EGFR
or PDK1 synergistically inhibited spheroid growth in combination with PI3K alpha inhibition. Our work
therefore supports further investigation into combined PI3K and RTK inhibition for the treatment of pan-
creatic cancer.

32. A predictive model of “on-target” drug resistance (for poster)

1,3
2
1
Daniel O’Neill , Maryam Clausen , Mike Firth , Jonathan Wrigley , Maria Emanuela Cuomo 1
1
1 AstraZeneca,IMED Discovery Sciences, Cambridge, UK

2 AstraZeneca, IMED Discovery Sciences, Gothenburg, SE
3 AstraZeneca, IMED Oncology, Cambridge, UK
Acquired drug resistance represents a major challenge in the development of therapeutic agents for the
treatment of cancer. The problem is exemplified by non-small cell lung cancer (NSCLC) containing activat-
ing EGFR mutations. NSCLC tumours driven by EGFR variants are initially sensitive to tyrosine kinase
inhibitors (TKI), but some patients show renewed disease progression through expansion of tumour cell
clones harbouring additional EGFR mutations. Models of drug resistance are therefore necessary to in-
crease our understanding of resistance mechanisms, predict variants that will arise in patients and improve
drug development strategies. In the past, this problem has been addressed by screening large panels of gene
variants for drug resistance phenotypes in cell culture. Previous methods such as chemical mutagenesis
has the advantage of testing mutations genome wide but did not cover all possible amino acid variants
in a specific region of interest. On the other hand, saturation mutagenesis with “on target” cDNA librar-
ies resulted in expression of all possible variants but in a non-physiological setting, detached from their
endogenous regulatory mechanisms. For the first time, CRISPR/Cas9 allows endogenous modification of
genes and facilitates introduction of all possible mutations at a specific locus, whilst maintaining endog-
enous gene architecture, chromosomal context and epigenetic mileu. Here we describe the novel CRISPR-
based methodology targeted endogenous mapping of pharmacological resistance developed to identify “on
target” mutations in key oncogenes that drive pharmacological resistance. Using EGFR as a target, we show
how directed evolution of some residues in the EGFR kinase domain can be predictive of the resistance
observed in the clinic in response to known TKIs, such as gefitinib and osimertinib. In fact, in the region
analysed, we can recapitulate the variants observed clinically. We also identify amino acids chemically and
structurally able to confer resistance to a TKI in structurally conserved tyrosine kinases such as BCR-Abl
and Kit.