Activation of CRLF2/IL7RA Signaling in Normal Human Cord Blood Hematopoietic Progenitors Induces Philadelphia-like B-Cell Precursor Pre-Leukemia and Leukemia In Vivo
Authors: Geron Ifat , Savino Angela Maria , Tal Noa , Brown John , Turati Virginia A , Lee Yu Nee , James Chela , Rein Gil Avigail , Birger Yehudit , Muler Inna , Hameiri Michal , Marcu-Malina Victoria , Fischer Ute , Borkhardt Arndt , Kirsch Ilan M. , Nagler Arnon , Enver Tariq , Izraeli Shai 
Published: October 2019

Philadelphia-like (Ph-like) B-cell precursor acute lymphoblastic leukemia (BCP-ALL) is a subgroup of BCP-ALL with an expression pattern similar to BCR-ABL+ BCP-ALL that is associated with poor prognosis.

Aberrant expression of CRLF2 in BCP-ALL constitutes the majority of Ph-like BCP-ALL cases. CRLF2 is a receptor subunit that together with the IL7RA subunit comprises the receptor of the proinflammatory cytokine TSLP. Though activation of the IL7R pathway is commonly associated with T-cell malignancies, we previously described IL7RA-activating mutations in BCP-ALL predominantly in the context of CRLF2 aberrant expression (Shochat C. et al. J. Exp. Med. 2011).

Here we aimed to test the role of aberrations in CRLF2 and IL7RA in the development of Ph-Like BCP-ALL. Both CRLF2 and TSLP differ extensively between mice and human in amino acid sequence and in lineage expression pattern; loss-of-function germline mutations in IL7RA are associated with lack of B and T cells in mice but with lack of only T cells in humans. Hence, we chose to test the hypothesis that activation of CRLF2/IL7RA contributes to the development of Ph-like BCP-ALL in the context of human lymphopoiesis by using a human xenograft system.

To aberrantly activate TSLP/IL7 signaling, we transduced cord-blood (CB) CD34+ hematopoietic progenitors with a set of lentiviral vectors carrying CRLF2 and/or IL7RA [(wild type (IL7RAwt) or IL7R bearing an activating mutation (IL7RAins)] under a B-cell promoter/enhancer (to accentuate B-cell lineage expression). The backbone vector (BB) expressing GFP was used as a control. Transduced CB cells were transplanted into NOD/LtSz-scid IL2Rγnull (NSG) mice and engrafted cells were analyzed 24-30 weeks after transplantation. To test for self-renewal capacity, BM cells from primary engrafted mice were serially transplanted into secondary recipients and the occurrence of human engraftment was tested 24-30 weeks after transplantation.

Enforced expression of activated IL7RA with or without CRLF2 led to a significant block in B-cell development at the B-cell progenitor stage (CD19+CD10+sIgM-) in vivo resembling the differentiation stage of leukemic cells (figure Bi). Repertoire sequencing of CD10+CD19+-transduced cells that were sorted from BM of transplanted mice revealed a significantly higher population of DJ-rearranged cells in the CRLF2-IL7RAins-transduced population than in BB-transduced cells (mean ratio of DJ/total rearrangement: BB:0.35+/-0.024, CRLF2-IL7RAins:0.76+/- 0.07, p=0.039, n=3 paired cord blood), in agreement with the early differentiation block phenotype measured by immunophenotyping. These cells furthermore exhibited a Ph-like gene expression pattern when compared to BB-transduced cells in gene set enrichment analysis.

Overexpression of IL7RA alone significantly enhanced the early-B fraction (CD19+CD10+CD34+) in the BM of transplanted mice (figure Bii). Additionally, aberrant expression of IL7RA enhanced self-renewal capacity as was evident by an increased ability of the transduced cells to engraft in secondary recipients (number of mice with detectable human engraftment out of secondary transplanted mice: BB:0/6, CRLF2-IL7RAwt:0/3, CRLF2-IL7RAinst:0/3, IL7RAwt-GFP:3/6, IL7RAins-GFP:5/8).

Notably, in one case, secondary transplantation of IL7RAins-transduced CB triggered the development of acute BCP-ALL. The leukemic cells (CD19+CD10+CD34+sIgM-) were clonal as validated by V(D)J rearrangement (figure Cii), had the ability to further propagate in serial transplantations and gained secondary Ph-like BCP-ALL-characteristic chromosomal deletions in the short arm of chromosome 9 (in the region including the genes for CDKN2A/B, PAX5 and JAK2) and the short arm of chromosome 7 (the region including IKZF1) (figure Ciii).

These results support the hypothesis that aberrant activation of the CRLF2/IL7RA pathway in human B-cell lineage progenitors creates a pre-leukemic state by arresting differentiation of B-cell progenitors, instating Ph-like expression pattern and inducing self-renewal. This is the first model of de novo Ph-like BCP-ALL development from normal human hematopoietic progenitors in vivo. Additionally, we present here a first direct in vivo demonstration of a role for IL7 in human B-cell development.

Disclosures

No relevant conflicts of interest to declare.