Abstract 3357: Identifying mechanisms of acquired resistance to lorlatinib utilizing a genome-wide CRISPR-Cas9 screen
Authors: Kalna Joshua Renn , Matkar Smita , O'Drisoll Emily , Balyasny Skye , Calafatti Matteo , Gerelus Mark , Groff Dave , Acholla Tina , Casey Colleen E. , Kamitsuka Paul , Li Grant , Pastor Steven J. , Witek Gabriela , Krytska Kateryna , Lindsay Jarrett , Shalem Ophir , Mossé Yael P. 
Published: March 2024

Abstract
Survival rates for neuroblastoma (NB), a predominantly pediatric solid tumor arising from the sympathoadrenal neural crest, have improved incrementally from 25% to 50% over three decades with dramatic escalation in therapy intensity. Gain-of-function mutations in anaplastic lymphoma kinase (ALK), of which 85% occur at residues F1174, F1245, and R1275, confer inferior survival compared to patients with wild-type ALK. Lorlatinib, a third generation ALK inhibitor, has completed Phase 1 testing in the NANT Consortium and has moved to frontline therapy in the Children’s Oncology Group. The robust and sustained activity observed with minimal toxicity is transient in relapse patients harboring MYCN amplification and ALK activation. We aim to elucidate and target mechanisms of acquired resistance that emerge from lorlatinib treatment. We utilized the Brunello library, a genome-wide CRISPR-CAS9 loss of function screen targeting 19,114 genes (4 sgRNA per gene), which allows for a high confidence of detection of hits. We screened four clinically representative cell lines: Kelly (ALKF1174L, MYCN-amplified, p53P117T), SMS-SAN (ALKF1174L, MYCN-amplified), LAN5 (ALKR1275Q, MYCN-amplified), and SY5Y (ALKF1174L, MYCN-nonamplified). sgRNAs were ranked according to the degree of depletion or enrichment. We validated top depleted targets through cell-based assays and immunoblotting. We identified several actionable targets across multiple NB cell lines that, when depleted, increase the efficacy of lorlatinib, including FGFR2, BCL2L1, SOX11, SMARCA2, and SMARCD2. Genetic knockout of FGFR2 led to a 40% reduction (p&lt;0.05) in KO cells upon lorlatinib treatment compared to control. PROTAC degradation of the anti-apoptotic BCL-xL (BCL2L1) revealed the combination of lorlatinib and the PROTAC led to a decrease in IC50 in vitro. Additionally, we identified conserved core components of the SWI/SNF complex, a subfamily of ATP-dependent chromatin remodelers, as well as SOX11, a regulator of the SWI/SNF complex and transcription factor of the core regulatory circuitry in adrenergic high-risk NB. In conclusion, we identified actionable targets across four NB cell lines that, when depleted, increase the efficacy of lorlatinib. Depletion of FGFR2 led to increased sensitivity to lorlatinib, however enzymatic inhibition showed minimal effect. Future investigation into dual targeting of ALK and FGFR2 through novel alternative methods is warranted. Furthermore, we observed a synergistic relationship between lorlatinib and a BCL-xL PROTAC in vitro. Ongoing in vivo studies will test the combinatorial efficacy in lorlatinib-naïve and resistant models. Finally, we are investigating epigenetic remodeling by the SWI/SNF complex and its regulators in NB cells during lorlatinib treatment through targeted genetic manipulation of SOX11 to understand its role as a master transcriptional regulator of MYCN and FGFR2.
Citation Format: Joshua Renn Kalna, Smita Matkar, Emily O'Drisoll, Skye Balyasny, Matteo Calafatti, Mark Gerelus, Dave Groff, Tina Acholla, Colleen E. Casey, Paul Kamitsuka, Grant Li, Steven J. Pastor, Gabriela Witek, Kateryna Krytska, Jarrett Lindsay, Ophir Shalem, Yael P. Mossé. Identifying mechanisms of acquired resistance to lorlatinib utilizing a genome-wide CRISPR-Cas9 screen [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3357.