Abstract LB251: Blocking genomic instability delays acquired resistance to MAPK inhibitor therapy in melanoma
Authors: Dharanipragada Prashanthi , Zhang Xiao , Liu Sixue , Lomeli Shirley H. , Hong Aayoung , Wang Yan , Yang Zhentao , Vega-Crespo Agustin , Ribas Antoni , Moschos Stergios J. , Moriceau Gatien , Lo Roger S. 
Published: April 2023

Abstract
Background: In cutaneous melanoma, the burden of chromothripsis is high prior to targeted therapy, and additional chromothripsis appears to be a key evolutionary mechanism by which cancer rapidly generates and accumulates highly dynamic structural variants (SVs). Blocking cancer genomic instability may prevent tumor escape from targeted therapies.
Methods: We assembled three cohorts of tissues for WGS-based analysis of SVs. The first cohort consisted of patient-matched normal tissues, BRAF V600MUT melanoma tumors before MAPKi therapy and at disease progression (n=10 normal tissues; n=10 pretreatment tumors; n=17 acquired-resistant tumors; n=10 patients). The second cohort consisted of rapid autopsy melanoma tissues (n=3 normal tissues; n=12 acquired-resistant tumors; n=6 metastatic organ sites). The third cohort consisted of cutaneous PDX tumors. To study acquired MAPKi-resistance at the whole-genome level, we subjected PDXs (n=6 models; 1 BRAF MUT and 5 NRAS MUT models) to MAPKi therapy in NSG mice at doses sufficient to elicit tumor regression, and then generated acquired MAPKi-resistant tumors. In total, we used vehicle-treated tumors (n=6), acquired-resistant tumors (n=12), and patient-matched normal tissues (n=6) to generate WGS data.
Results: Analysis of genomic amplicons due to intrachromosomal complex genomic rearrangements (CGRs) and extrachromosomal circular DNAs (ecDNAs) uncovered a significant (unpaired Student’s t-test, p=0.0002) association between acquired-resistant tumors and CGRs and/or ecDNAs harboring bona fide MAPKi-resistance genes and revealed copy number amplification of BRAF (range 4.5-27), NRAS (range 5-13), HRAS (range 13-16), MYC (range 12-15) and EGFR (CN 4.6-5), known to drive acquired MAPKi-resistance. Moreover, we validated a recurrent ecDNA by direct isolation and high-depth sequencing using a new approach referred to as CRISPR-CATCH. This alternative technique confirmed the circularized junctions of a 890 kb, driver ecDNA within this acquired-resistant clinical tumor sample. Additionally, resistance-specific (versus sensitivity-specific) chromothriptic single-base substitutions (SBSs) enriched for signatures of defects in base excision repair (BER) and in DNA mismatch repair (MMR) (Wilcoxon rank sum test, p=0.04 and p=0.005 respectively) in 14 of 31 resistant tumors (10 of 16 patients). Moreover, breakpoint-sequence analysis inferred non-homologous end-joining (NHEJ) as critical, and homologous recombination repair (HRR) as adjunctive, to DNA double-stranded break repair underlying CGR and ecDNA formation harboring MAPK-reactivation or MAPKi resistance-driver genes. Inhibition of DNA-PKc or PARP1/2, even only initially during MAPKi treatment, suppressed acquired MAPKi-resistance in melanoma cell lines (BRAF V600MUT n=3 and NRAS Q61MUT n=3) and blunted the expansion of ecDNA + CGR genomic spans. In vivo, DNA-PKi in combination with MAPKi forestalled resistance, reduced ecDNA and CGRs size, and suppressed the contribution of NHEJ in 5 out of 5 cutaneous melanoma PDXs analyzed.
Conclusions: Our findings advance the concepts that preventing—instead of reversing—acquired resistant phenotypes may be more clinically impactful and that targeting DNA-PKCS and NHEJ lies at the center of this approach in stabilizing cancer genomes during oncogene-targeted therapies.
Citation Format: Prashanthi Dharanipragada, Xiao Zhang, Sixue Liu, Shirley H. Lomeli, Aayoung Hong, Yan Wang, Zhentao Yang, Agustin Vega-Crespo, Antoni Ribas, Stergios J. Moschos, Gatien Moriceau, Roger S. Lo. Blocking genomic instability delays acquired resistance to MAPK inhibitor therapy in melanoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr LB251.