E-Poster Presentation 34th Lorne Cancer Conference 2022

HMGN1 expression predisposes Down Syndrome patients to develop P2RY8-CRLF2 acute lymphoblastic leukaemia (#342)

Elyse Page 1 , Susan Heatley 1 , Jacqueline Rehn 1 , David Yeung 1 , Paul Thomas 1 , Deborah White 1
  1. SAHMRI, Adelaide, SA, Australia

Introduction

Children with Down Syndrome (DS) acute lymphoblastic leukemia (ALL) have increased relapse rates, chemotherapy related toxicity and poor overall survival compared to non-DS patients. The P2RY8-CRLF2 gene fusion has been identified in 60% of DS-ALL patients, yet the genomic basis for the predisposition remains unknown. The high mobility-group nucleosome-binding protein 1 (HMGN1) on chromosome 21 may play a role in DS-ALL leukemogenesis.

 

Methods

mRNA-seq was performed on ALL patient blasts. Fusion transcripts were identified and mRNA expression data was generated. CRISPR/Cas9 gRNAs were designed targeting P2RY8 and CRLF2 in Jurkat cells ± HMGN1 to create the 320 KB deletion (PAR1) resulting in P2RY8-CRLF2. Cells were single-cell sorted for TSLPR (CRLF2/IL-7Ra) and phosphorylation of signalling proteins were measured.

 

Results

Significantly higher HMGN1 expression was identified in P2RY8-CRLF2 ALL patients, compared to the control (BCR-ABL1+; p<0.0001). To understand the role of HMGN1 in P2RY8-CRLF2 development, HMGN1 was overexpressed (1.5-fold; HMGN1H) in Jurkat cells to represent a trisomy expression level prior to inducing the gRNAs with undirected repair. Gene editing activity, TSLPR and CRLF2 mRNA expression were higher in P2RY8-CRLF2 + HMGN1H cells compared to P2RY8-CRLF2-only cells (p=0.034 and <0.001 respectively). This suggests higher HMGN1 expression predisposed to P2RY8-CRLF2 formation after a double-stranded DNA break (DSB), consistent with HMGN1H observed in patients with P2RY8-CRLF2. HMGN1H cells also demonstrated significantly increased pSTAT5 (MFI: 2359±1), pAKT (MFI: 2339±6) and pERK (MFI: 2478±48) compared to P2RY8-CRLF2-only cells (MFI: pSTAT5:1910±10; pAKT:1727±14; pERK:1946±6; all p<0.001), confirming cooperation between CRLF2 and HMGN1.

 

Conclusion

We demonstrate that P2RY8-CRLF2 is associated with high expression of HMGN1 in ALL patient cells. Furthermore, we identify HMGN1H cells favour PAR1 deletion and the subsequent formation of the P2RY8-CRLF2 gene fusion after DSB and increase cell signalling pathways. Understanding the role of HMGN1 in DS-ALL patients has the potential to lead to novel therapeutic interventions in this high-risk group of patients with poor efficacious therapeutic options.