HMGN1 and P2RY8-CRLF2 cooperate in Down Syndrome acute lymphoblastic leukemia

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Cytokine receptor like factor 2 (CRLF2) is dysregulated in approximately 50% of high-risk and 60% of Down Syndrome (DS) acute lymphoblastic leukemia (ALL) patients(1). Dysregulation of CRLF2 results in the upregulation of thymic stromal lymphopoietin receptor (TSLPR) and JAK/STAT, PI3K and Ras signalling pathways and is associated with poor survival outcomes. CRLF2 is most commonly rearranged in DS-ALL patients via a 320 KB deletion in the pseudoautosomal region of the X/Y chromosome resulting in the P2RY8-CRLF2 gene fusion, or can become mutated; CRLF2 p.F232C. The increased frequency of the P2RY8-CRLF2 fusion in DS-ALL patients indicates a predisposition to the development of this fusion, however, the genetic basis is unknown and warrants further investigation.

Genes located in the DS critical region of chromosome 21, such as HMGN1, have been implicated in the development of ALL in DS patients(2-4). Here, a CRISPR/Cas9 KO of the chromosome 21 gene, HMGN1, in a trisomy 21 leukemic cell line (SET-2) and xenograft has been generated to develop a deeper understanding of the role of HMGN1 in CRLF2 driven DS-ALL development and progression.

HMGN1 has a central role in cell-cycle and transcriptional events(5, 6) and here, using an in vivo HMGN1 KO and control JAK2 KO, the important role of HMGN1 in both proliferation and survival pathways of trisomy 21 SET-2 CRLF2 p.F232C cells were confirmed. We demonstrated HMGN1 KO mitigated leukemic phenotypes including hepatosplenomegaly, thrombocytopenia, and anemia, commonly observed in leukemia patients, and significantly increased survival in vivo. This indicates that HMGN1 has driver potential in DS-ALL. Significantly, HMGN1 overexpression occurs due to trisomy 21, suggesting DS patients who acquire P2RY8-CRLF2 may not require a ‘second hit’ for leukemic transformation, explaining the increased frequency of P2RY8-CRLF2 in DS-ALL patients.

Using in vitro Ba/F3 and murine haematopoietic stem cell P2RY8-CRLF2 models, the cooperation between P2RY8-CRLF2 and HMGN1was also confirmed. Leukemic transformation was achieved via the co-expression of P2RY8-CRLF2 and HMGN1 in Ba/F3 or murine stem cells. Although overexpression of HMGN1 or P2RY8-CRLF2 alone in Ba/F3 and murine stem cells is non-transforming, co-expression of HMGN1 with the P2RY8-CRLF2 gene fusion or WT CRLF2 leads to cytokine independence and altered methylation and signaling profiles. Furthermore, an upregulation of cell signalling pathways was observed when P2RY8-CRLF2 and HMGN1 were co-expressed. We identify enhanced STAT5 expression in HMGN1 and P2RY8-CRLF2 co-expressing cells and demonstrate a feedback loop resulting in increased CRLF2 expression and cell signaling. Therefore, high HMGN1 expression allows the signaling threshold of P2RY8-CRLF2 cells to be overcome, enabling leukemic transformation. Similar mechanisms have recently been demonstrated by HOXA9 co-occupying the same sites as STAT5 to overcome the signaling threshold required for T-ALL transformation(7). Interestingly, HMGN1 has recently been identified to associate with HOXA cluster genes which further activates STAT5(8) supporting the proposed mechanism.

A synergistic combination therapy comprising the JAK2 inhibitor, fedratinib, and de-methylase inhibitor, GSK-J4, was identified to target P2RY8-CRLF2 and HMGN1 co-expressing cells, however, CRLF2 p.F232C cells were less sensitive to this therapy and activated different signalling pathways. This synergistic drug combination was validated in clinical samples, and is particularly important, not only for DS-ALL patients, but also patients harboring +21 or intrachromosomal amplification of chromosome 21 (iAMP21) as HMGN1 is also highly expressed in these subtypes.

The identification of P2RY8-CRLF2 and HMGN1 cooperation could positively affect the treatment of patients with DS-ALL, CRLF2r or +21 by targeting HMGN1, and lead to better treatment outcomes for patients who currently have poor overall survival. Furthermore, the mechanism of leukemic transformation in P2RY8-CRLF2 and HMGN1 co-expressing cells was identified in both the Ba/F3 model and murine stem cell model via increased CRLF2, TSLPR and cell signalling pathways.

In summary, this study provides critical insight into the development and persistence of CRLF2 driven DS-ALL using in vitro and in vivoxenograft models. For the first time, the important role of HMGN1 in the proliferation and survival of DS-ALL cells and cooperation with P2RY8-CRLF2 for increased cell signalling has been identified.  These findings suggest HMGN1 is a potential target for a precision treatment approach in DS-ALL. A synergistic combination therapy targeting CRLF2 and HMGN1 co-expressing cells, has been investigated. These findings assess the leukemic potential of HMGN1 and provide significant understanding of its cooperation with P2RY8-CRLF2. Together, these data provide critical insight into the development and persistence of CRLF2r DS-ALL and identify HMGN1 as a potential therapeutic target. The outcomes from this study present an opportunity to reduce the toxicity DS-ALL patients experience from current treatment regimens and improve outcomes in this high-risk group of patients.


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Elyse Page

Post-doctoral research fellow, South Australian Health and Medical Research Institute

Elyse received her PhD from the University of Adelaide in 2021 and was awarded the highly prestigious Dean’s Commendation for Doctoral Thesis Excellence. She is currently a post-doctoral researcher with the leukaemia research group at SAHMRI and a member of the Australian Society for Medical Research committee. Elyse has been awarded the Simone Family Fellowship to continue her research investigating new driver genes and finding novel therapies for children with acute lymphoblastic leukaemia.