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Page 288 Cancer Drug Resist 2018;1:266-302 I http://dx.doi.org/10.20517/cdr.2018.18

Acquired-resistance to anti-EGFR monoclonal antibodies (mAbs) is a major cause of treatment failure in
patients with colorectal cancer (CRC), highlighting the need for more effective therapeutic strategies in
such patients. We have previously shown that acquired-resistance to anti-EGFR mAb ICR62 in DiFi CRC
cells (DiFi62) is accompanied by an increased EGFR expression, and augmented HER-2/HER-3 signalling
and sensitivity to the pan-HER blocker afatinib. In this study, we investigated the therapeutic potential
of the CDK inhibitors (palbociclib and ribociclib) as a single agent or in combination with the IGF-IR
inhibitors (NVP-AEW541, NVP-ADW742) or MEK inhibitor (trametinib) in parental DiFi cells and their
resistant varaints. The ICR62 resistant varaint was found to be highly resistant to treatment with palbo-
ciclib and trametinib. Interestingly, the gefitinib resistant DiFiG cells were found to be sensitive to NVP-
AEW541, NVP-ADW742, ribociclib, palbociclib and trametinib. While the combination of CDK inhibitors
with NVP-AEW541 yielded synergistic growth inhibition in DiFi62 cells, the same combination was an-
tagonistic in DiFiG cells. In addition, trametinib combined with either ribociclib or NVP-AEW541 yielded
synergistic effect in both DiFi62 and DiFiG variants. Using proteome profiler kits, we found DiFiG cells
had an increased phosphorylation of EGFR, insulin receptor, IGF-IR and ERK1/2, but HER-2 and HER-3
were undetectable. A significant increase in the levels of CDK4/6 was observed in DiFi62, but its level of ex-
pression was lower in the DiFiG variant. Moreover, treatment with CDK inhibitors or trametinib was able
to entirely block the signalling of CDK4/6 and cyclin-D1 in DiFiG cells, but not in DiFi62 cells, suggesting
that the mechanisms of acquired resistance to anti-EGFR mAb or TKI in these two variants are indeed dif-
ferent. Taken together, our results support further investigation on the therapeutic potential of combining
CDK4/6 and IGF-IR inhibitors in CRC cells with acquired resistance to anti-EGFR mAb.

41. ATP-binding cassette - transporter upregulation; a potential mechanism of resistance to
CDK7 inhibitors THZ1 and ICEC0942

Georgina P. Sava, Charles Coombes, Laki Buluwela, Simak Ali

Division of Cancer, Department of Surgery & Cancer, Imperial College London, Hammersmith Hospital
Campus, London, UK

Cyclin-dependent kinase (CDK)7 is necessary for transcription initiation, via phosphorylation of the C-
terminal domain of RNA polymerase II, and for cell cycle progression, through T-loop phosphorylation
of the cell cycle CDKs. Recently it has been shown that CDK7 inhibition may be a useful novel approach
in the treatment of multiple cancer types. We have developed a new, orally bioavailable selective CDK7
inhibitor, ICEC0942, that is currently being evaluated in a Phase I clinical trial for advanced solid malig-
nancies. Drug resistance is currently a major problem facing cancer treatment and cancer cells are able to
evade therapy via a multitude of mechanisms, therefore we sought to identify mechanisms by which can-
cer cells may become resistant to CDK7 inhibition. Through long-term drug exposure, we have developed
MCF7 breast cancer cell lines that are resistant to both ICEC0942 and the covalent CDK7 inhibitor, THZ1.
Importantly, whilst cells exposed to ICEC0942 develop resistance to both ICEC0942 and THZ1, cells cul-
tured long-term with THZ1 retain sensitivity to ICEC0942. We have identified upregulation of multi-drug
resistance ATP-binding cassette (ABC) transporters in the resistant cell lines. Specifically, the ICEC0942-
and THZ1-resistant cells have upregulation of ABC subfamily B member 1 (ABCB1) or MDR1 and ABC
subfamily G member 2 (ABCG2) or BCRP, respectively. Increased ABC-transporter mediated efflux activ-
ity was confirmed using flow cytometry and verapamil and novobiocin, specific inhibitors of ABCB1 and
ABCG2, were able to rescue CDK7 inhibitor sensitivity in the relevant resistant cell lines, demonstrating
the importance of ABC-transporter activity in these resistance models. In conclusion, we have identified
ABCB1 upregulation as a common mechanism of resistance to ICEC0942 and THZ1, and ABCG2 upregu-
lation as a mechanism of resistance to THZ1. Overall, we hope that identifying potential mechanisms of
CDK7 inhibitor resistance will help guide their future clinical use.