Abstract 4761: Identifying molecular targets to overcome CNS infiltration in acute lymphoblastic leukemia associated with an activating mutation of the NSD2 histone methyltransferase
Authors: Kaestner Charlotte L. , Sobh Amin , Li Jianping , Raburn Katelyn , Riva Alberto , Brant Jason O. , Bennett Richard L. , Licht Jonathan D. 
Published: April 2023

Background: One of the major clinical challenges in acute lymphoblastic leukemia (ALL) is the treatment of central nervous system (CNS) involvement. CNS-directed therapy is currently limited to agents associated with substantial neurotoxicity and the lack of mechanistic understanding of how ALL cells infiltrate the CNS is preventing novel therapy development. Our laboratory has previously shown in murine xenografts that ALL cells with a point mutation (E1099K) in the histone methyltransferase NSD2 aggressively infiltrate not only the leptomeninges of the brain, but also the brain parenchyma. Accordingly, we also showed that NSD2-E1099K cells have an enhanced ability to migrate through a Boyden chamber and adhere to endothelial cells of the blood brain barrier (BBB cells). Furthermore, RNA-seq data on four NSD2-E1099K cell lines revealed genes that may play a role in ALL brain infiltration. However, it remains unknown which of the genes upregulated by NSD2 could be potential therapeutic targets against CNS leukemia.

Aim: This study aims to identify therapeutically targetable genes that are important for (1) migration of ALL cells through a Boyden chamber, (2) adhesion of ALL cells to BBB cells, and (3) intercellular signaling between ALL and BBB cells.

Methods: We used a focused CRISPR-gene-knockout library targeting 500 genes upregulated in NSD2-E1099K cells to ascertain genes important for migration in the RCHACV cell line. Next, we used the genome-wide Brunello library to identify genes critical for ALL cell adhesion to BBB cells. To study intercellular communication between ALL and BBB cells, we co-cultured SEM-WT or SEM-NSD2-E1099K cells with BBB cells for 48h. ALL cells were then isolated from BBB cells using CD19+ magnetic beads and all groups were further processed for RNA-seq.

Results: Our CRISPR screen studies identified genes whose knockout led to enhancement of migration or adhesion, as well as genes whose knockout resulted in inhibition of migration or adhesion. One of the top candidate genes identified in the migration screen was PTPRG. Validation studies with shRNA and CRISPRa constructs confirmed that PTPRG is a modulator of migration and adhesion in NSD2-E1099K ALL cells. In addition, RNA-seq studies identified important differences in how SEM-WT (low-CNS infiltrating) and SEM-NSD2-E1099K (high-CNS infiltrating) cells communicate with BBB cells. Specifically, our analysis predicted that IL1B secretion of SEM-NSD2-E1099K cells, but not SEM-WT cells, leads to increased IL1B target gene expression in BBB cells, potentially contributing to CNS infiltration.

Conclusion: Our findings implicate PTPRG and the IL1B pathway as important modulators of migration and adhesion or intercellular signaling, respectively. These genes may have the potential to be novel therapeutic target for preventing ALL brain infiltration.

Citation Format: Charlotte L. Kaestner, Amin Sobh, Jianping Li, Katelyn Raburn, Alberto Riva, Jason O. Brant, Richard L. Bennett, Jonathan D. Licht. Identifying molecular targets to overcome CNS infiltration in acute lymphoblastic leukemia associated with an activating mutation of the NSD2 histone methyltransferase. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4761.