Identification of determinants of sensitivity to duocarmycin analogues: A class of potent anti-cancer drugs

Duocarmycin analogues are currently in pre-clinical development as hypoxia-activated prodrugs and as so-called payloads for antibody-drug conjugates for the treatment of cancer. Although the mechanism of action (MOA) of duocarmycins has been elucidated as DNA minor groove alkylation, not much is known about the factors involved in the intrinsic sensitivity of cancer cells to duocarmycins. This thesis aims to identify the determinants of sensitivity of duocarmycin analogues by utilising three independent approaches. First, a hypothesis-driven approach was based on literature that suggested an alternative mechanism of action of duocarmycin analogues where ALDH1A1 alkylation was proposed as the main target of duocarmycin analogues rather than DNA alkylation. To test ALDH1A1 as a potential determinant of sensitivity, a homozygous ALDH1A1 knockout of the A549 cell line was established by using dual CRISPR-Cas9 ribonucleoproteins. The effect of ALDH1A1 knockout on the cytotoxicity of duocarmycin analogues was tested by anti-proliferative and anti-clonogenic assays. The results showed that ALDH1A1 does not play a key role in the MOA of these duocarmycin analogues and thus the original proposed MOA of DNA alkylation was explored, demonstrating that duocarmycin analogues elicit RAD51 foci formation in A549 cells. Moreover, homologous recombination (HR) deficient cell lines (BRCA2-/- in human colorectal cancer cell line and RAD51D-/- in CHO hamster cell line) showed hypersensitivity to treatment with duocarmycin analogues. The findings suggested that DNA alkylation is the main MOA and homologous recombination repair proteins are potential determinants of sensitivity to duocarmycin analogues. The second approach aims to identify molecular pathways related to duocarmycin analogues sensitivity by correlating phenotypic cytotoxicity data to whole-genome transcriptomic data. This was achieved by utilising basal gene expression levels in two carcinoma cell line panels. The basal gene expression levels data was correlated to anti-proliferation cytotoxic potency IC50 values which is a measure of drug concentration causing 50% inhibition in cell proliferation in contrast to control. The results suggested DNA repair (DNA recombination), cell cycle regulation (G1-S phase transition), and RNA processing (mRNA transcription regulation) as key processes related to sensitivity to duocarmycin analogues. Differential gene expression analysis between sensitive and resistant cell lines as defined by the IC50 data was also carried out identifying RAD54 and FANCD2 to be upregulated in sensitive cell lines. To determine potential single genomic determinants of sensitivity to duocarmycins, genome-wide knockout CRISPR-Cas9 screens were carried out in two cell lines, UT-SCC-54C and KBM7, and with two duocarmycin analogues. Amongst the most significant gene hits were RAD18, RAD54L, SWSAP1, and ZSWIM7 that function in HR pathway. Other highly significant genes were UVSSA and ERC88 that function in transcription-coupled nucleotide excision repair and POLK and PRIMPOL that function in the translesion synthesis pathway. Moreover, knockout mutations of mTORC1 regulatory proteins such as tuberous sclerosis complex (TSC1 and TSC2) and STK11 were identified as potential markers to duocarmycin analogues cytotoxicity.

Name thesis:degree_name

PhD

Level thesis:degree_level

Doctoral

Discipline thesis:degree_discipline

Biomedical Science

Grantor dc:publisher

ResearchSpace@Auckland

Year dc:date.issued

2020

Author dc:creator

• Ghamri, Nour

Advisors dc:contributor.advisor

• Pruijn, Frederik
• Bohlander, Stefan
• Hunter, Francis
• Lee, Tet-woo

Statement dc:rights

• Items in ResearchSpace are protected by copyright, with all rights reserved, unless otherwise indicated.

Licence dc:rights.uri

• https://researchspace.auckland.ac.nz/docs/uoa-docs/rights.htm

Handle dc:identifier.uri

https://hdl.handle.net/2292/56941

OAI identifier oai:identifier

oai:researchspace.auckland.ac.nz:2292/56941