CRLF2 Rearrangement Is Not Associated with Activated mTOR Pathway in NOD/SCID/huALL Xenograft Mouse Model.
Authors: Hasan Md. Nabiul , Bresolin Silvia , Bronzini Ilaria , Palmi Chiara , Queudeville Manon , Eckhoff Sarah Mirjam , Trentin Luca , Cazzaniga Giovanni , te Kronnie Geertury , Debatin Klaus-Michael , Meyer Lüder-Hinrich 
Published: October 2019

Abstract
Abstract 2518
B cell precursor acute lymphoblastic leukemia (BCP-ALL) is a heterogeneous disease and frequently associated with genetic alterations. However about one quarter of ALL patients lack characteristic chromosomal rearrangements representing a subset of leukemia not well understood. Expression of cytokine receptor-like factor 2 (CRLF2) has recently been shown to be up-regulated as well as mutated in BCP-ALL patients, including Down syndrome (DS-ALL) patients, lacking recurring chromosomal translocations. Several alterations in CRLF2 resulting in its overexpression have been described: a focal ∼320 kilobase interstitial deletion of the pseudo-autosomal region of the sex chromosomes (Xp22.23 or Yp11.32) that creates a fusion of CRLF2 to the G-protein-coupled purinergic receptor P2RY8 gene (P2RY8-CRLF2), translocation of CRLF2 with the IGH@ locus of chromosome 14, and a point mutation in CRLF2 resulting in a phenylalanine-to-cysteine substitution at amino acid 232 (F232C). Recently it has been shown that patients with CRLF2-P2RY8 fusion gene are associated with poor prognosis although they were initially stratified into intermediate risk (IR) group by minimal residual disease (MRD) criteria. This emphasizes the need to identify the mechanism of CRLF2 disregulation in leukemia thereby providing new therapeutic strategies.
We have characterized leukemia samples for the presence of CRLF2 expression at the protein level by FACS, at transcript level by RT-PCR, mutations in CRLF2 and JAK2 by next generation amplicon sequencing and CRLF2-P2RY8 fusion gene and IGH@ translocation by RT-PCR. Interestingly, in our recent study we have observed activated mTOR pathway in a subset of B-ALL xenograft leukemia without recurring genetic alterations that were sensitive to mTOR inhibition ex vivo and in vivo. In view of the recently suggested interconnection between TSLP/CRLF2 and PI3K/mTOR signaling networks we analyzed the mTOR pathway activation in CRLF2 rearranged ALL.
In this study we functionally address mTOR pathway activation in xenograft ALL samples with CRLF2 rearrangements and without any known chromosomal rearrangements ex vivo. We have adopted the NOD/SCID/huALL xenotransplant mouse model for this study. Xenograft leukemia cells with CRLF2 rearrangements and without recurring genetic alterations (CRLF2 rearrangements, n=4; CRLF2 wild type, n=11) were re-transplanted onto NOD/SCID mice and ALL cells were harvested at disease onset from leukemia bearing mice. The mTOR pathway activation was analyzed by flow cytometry assessing phosphorylation of ribosomal protein S6 (P-S6), a molecule downstream of the mTOR pathway. No significant difference in basal P-S6 level was found between CRLF2 rearranged and CRLF2 wild type leukemia. Moreover, reduction of the mTOR pathway activation upon mTOR inhibition by rapamycin and dual PI3K-mTOR inhibitor BEZ235 was analyzed. No significant reduction of the mTOR activity was observed in CRLF2 rearranged leukemia compared to CRLF2 wild type leukemia. Since JAK2 mutations have been associated with CRLF2 rearrangements, we also analyzed the JAK-STAT5 pathway assessing P-STAT5 ex vivo. Only one patient derived xenograft ALL sample carried a JAK2 mutation however no differential activation of JAK-STAT5 pathway was observed between JAK2wt/CRLF2r and JAK2mut/CRLF2r samples ex vivo. Also no significant difference in basal P-STAT5 was observed between CRLF2 rearranged and CRLF2 wild type leukemia. Additionally we checked the effect of mTOR inhibition on P-STAT5. No significant reduction in P-STAT5 level upon mTOR inhibition was observed pointing P-STAT5 activation independent of mTOR activation.
In this study we have shown that CRLF2 rearranged leukemia is not associated with activated mTOR pathway and also not sensitive to mTOR inhibition either by rapamycin or dual PI3K-mTOR inhibitor NVP-BEZ235. In CRLF2 rearranged and CRLF2 wild type leukemia JAK-STAT5 pathway did not show differential activation and was unaffected by mTOR inhibition. It remains to be clarified whether both pathways could be activated with appropriate stimuli as was demonstrated for diagnostic specimens. To fully understand the exact mechanism of CRLF2 rearranged leukemia development and providing new therapeutic strategies, study the interlinks with other cell-signaling pathways is mandatory.

Disclosures:
No relevant conflicts of interest to declare.