Exploring the cooperation and targetability of CRLF2 and HMGN1 in Down Syndrome Acute Lymphoblastic Leukaemia

Abstract

Cytokine receptor like factor 2 (CRLF2) is dysregulated in approximately 50% of highrisk and 60% of Down Syndrome (DS) acute lymphoblastic leukaemia (ALL) patients. 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. 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. Constituents of these signalling pathways are targetable with small molecule inhibitors. 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. Many groups have postulated the involvement of the high mobility nucleosome binding protein 1 (HMGN1) in DS-ALL, however, this body of work identifies its role and cooperation with P2RY8-CRLF2. Using in vivo and in vitro models, as well as novel CRISPR/Cas9 modelling, the role of HMGN1 in the development, proliferation and persistence of CRLF2 rearranged ALL is demonstrated here, for the first time. The trisomy 21 human xenograft HMGN1 knockout (KO) model presented here demonstrates that HMGN1 KO in CRLF2 p.F232C cells halts leukaemic progression in mice, reverses the leukaemic phenotype and increases murine survival outcomes. 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 leukaemic transformation. Using in vitro Ba/F3 and unique CRISPR/Cas9-generated P2RY8-CRLF2 models, the cooperation between P2RY8-CRLF2 and HMGN1 was also confirmed. Leukaemic transformation was achieved via the co-expression of P2RY8-CRLF2 and HMGN1 in Ba/F3 cells. To support this finding, modelling HMGN1 overexpression prior to generating the endogenous P2RY8-CRLF2 fusion with CRISPR/Cas9 increased the efficiency of fusion development. The identification of P2RY8-CRLF2 and HMGN1 cooperation could positively affect the treatment of patients with DS-ALL, CRLF2r or +21 and lead to better treatment outcomes for patients who currently have poor overall survival. Furthermore, the mechanism of leukaemic transformation in P2RY8-CRLF2 and HMGN1 co-expressing cells was identified in both the Ba/F3 model and CRISPR P2RY8-CRLF2 model via increased CRLF2 expression and an upregulation of TSLPR, as well as JAK/STAT signalling and increased gene activation marks via HMGN1 nucleosome remodelling. A synergistic combination therapy comprising the JAK2 inhibitor, fedratinib, and demethylase inhibitor, GSK-J4, was identified to target P2RY8-CRLF2 and HMGN1 coexpressing cells, however, CRLF2 p.F232C cells were less sensitive to this therapy and activated different signalling pathways. Furthermore, also presented here, is an additional combination therapy of fedratinib and the MEK inhibitor, selumetinib, to synergistically target cells harbouring the aggressive CRLF2 p.F232C mutation. In summary, this thesis provides critical insight into the development and persistence of CRLF2 rearranged DS-ALL. 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. Two synergistic combination therapies targeting CRLF2 and/or HMGN1 co-expressing cells, as well as an endogenous model of P2RY8-CRLF2 that provides a clinically relevant tool for identification of cooperating genes have been produced. Together, this body of work describes the leukaemic potential of HMGN1 and significant understanding of its cooperation with P2RY8-CRLF2. Findings from this thesis 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.