Therapies targeting somatic bystander genetic events represent a new avenue for cancer treatment. We recently identified a subset of colorectal cancer (CRC) patients who are heterozygous for a wild-type and a low activity allele (NAT2*6) but lack the wild-type allele in their tumors due to loss of heterozygosity (LOH) at 8p22. These tumors were sensitive to treatment with a cytotoxic substrate of NAT2 (6-(4-aminophenyl)-N-(3,4,5-trimethoxyphenyl)pyrazin-2-amine, APA), and pointed to NAT2 loss being a therapeutically exploitable vulnerability of CRC tumors. To better estimate the total number of treatable CRC patients, we here determined whether tumor cells retaining also other NAT2 low activity variants after LOH respond to APA treatment. The prevalent low activity alleles NAT2*5 and NAT2*14, but not NAT2*7, were found to be low metabolizers with high sensitivity to APA. By analysis of two different CRC patient cohorts, we detected heterozygosity for NAT2 alleles targetable by APA, along with allelic imbalances pointing to LOH, in similar to 24% of tumors. Finally, to haplotype the NAT2 locus in tumor and patient-matched normal samples in a clinical setting, we develop and demonstrate a long-read sequencing based assay. In total,>79.000 CRC patients per year fulfil genetic criteria for high sensitivity to a NAT2 LOH therapy and their eligibility can be assessed by clinical sequencing.

Treatment of advanced colorectal cancer remains challenging with only 20% of patients surviving 5 years after diagnosis. We previously demonstrated that loss of heterozygosity of NAT2 following 8p22 loss in colorectal cancer can result in collateral vulnerability that can be exploited therapeutically. Here, we evaluated 2841 known and novel anticancer agents and identified 43 showing increased toxicity towards cells with slow NAT2 activity. Potential mechanisms of action were identified by morphological profiling. Of these, 6 were demonstrated as novel direct substrates for NAT2. These findings are indicating towards further exploration of LOH-based treatment approach. 

The Arylamine-N-acetyltransferase-2 (NAT2) enzyme is involved in metabolism of commonly used drugs driving differences in efficacy and tolerability of treatments. To bridge the current knowledge gap on metabolism of cytotoxic drugs by NAT2, and identify anticancer agents whose effects depend on NAT2 activity, we assessed 147 clinically used drugs. Hit compounds were evaluated for metabolic conversion by acetylation in presence of recombinant NAT2. Among those 147 drugs we found doxorubicin, daunorubicin, epirubicin, valrubicin, teniposide, afatinib, carmustine, vincristine, panobinostat, and vorinostat to have increased toxicity to cancer cells expressing the rapid NAT2 allele. Additionally, we report NAT2-mediated acetylation of idarubicin, daunorubicin, doxorubicin, vorinostat, and CUDC-101. These findings have implications for pharmacogenomics and cancer precision medicine using conventional chemotherapeutic drugs, as improving their efficacy and safety may affect >4 million cancer patients worldwide that receive these drugs as standard of care.

Targeting tumor vulnerabilities such as synthetic and collateral lethality is a potential avenue for cancer chemotherapy. We suggest to exploit loss of heterozygosity (LOH) at highly polymorphic gene loci as a target for cancer therapy. Previously we have shown how 8p22 and 22q13 loss might be targeted in cancer when LOH in polymorphic gene in a cancer cell might make it sensitive to certain drug treatments compared to normal cells that remain heterozygous. 

Here, we used population genomics database analysis to enrich genes frequently lost in colorectal cancer (CRC) and other common tumor types (lung, breast, GI, GU cancers) with prevalent variants causing amino acid substitutions in or near active sites of enzymes. Through analysis of 3D protein structure models and literature review we identified 70 putative genes of interest that may be further investigated. We reproduce previously published functional assay CRISPR-Select for variant assessment in our laboratory and suggest it as a robust methodology for validation of variants of interest.