Abstract B21: A human-mouse xenograft model to evaluate therapies and study the role of TSLP-induced signals in Ph-like ALL
Authors: Francis Olivia , Su Ruijun , Martinez Shannalee , Baez Ineavely , Milford Terry-Ann , Bennett Terrence , Fisher Ross , Morris Christopher L. , Dovat Sinisa , Payne Kimberly J. 
Published: May 2015

Abstract
While the overall survival rate for children with B cell precursor acute lymphoblastic leukemia (B-ALL) is high, a subset of children with this disease are at high risk for relapse and death. Genome-wide analysis has shown that gene expression profiles in these high-risk B-ALLs is similar to that of Philadelphia chromosome–positive ALL and these are designated Ph-like ALL. Approximately half of Ph-like ALL are characterized by genetic defects resulting in overexpression of CRLF2. CRLF2, together with the IL-7Rα, forms a receptor complex that is activated by the cytokine, TSLP. The JAK-STAT5 pathway is phosphorylated downstream of this receptor complex activation. The activating JAK mutations found in some CRLF2 B-ALL have led to speculation that TSLP stimulation is not a factor in CRLF B-ALL. In preliminary studies to address this question we evaluated the effect of TSLP on a CRLF2 B-ALL cell lines with JAK defects and which have been reported to exhibit constitutive JAK-STAT5 activation. Our data show that TSLP increases STAT5 phosphorylation in these cell lines and also in primary CRLF2 B-ALL cells. Our next step was to evaluate the role of TSLP-CRLF2 interactions in vivo in the human-mouse xenograft model. However, mouse TSLP is different from most other cytokines produced in the xenograft in that it is species-specific and does not activate the human TSLP receptor complex that includes CRLF2. Thus, traditional xenograft models do not provide the TSLP-CRLF2 interactions that we believe to be a major factor in CRLF2 B-ALL. To overcome this obstacle we engineered immune-deficient NOD/SCID IL-2Rγ null (NSG) mice to express human TSLP (hTSLP+ mice) as well as control mice that lack the TSLP cytokine (hTSLP– mice). ELISA assays show serum hTSLP levels in the hTSLP+ mice that approximate the normal range in human serum. We used this hTSLP+/- xenograft model system to study the in vivo effects of TSLP on mice transplanted with Ph-like B-ALL. First, we used the hTSLP+/– xenograft model system to evaluate the in vivo effects of TSLP on survival and proliferation of transplanted CRLF2 B-ALL cells harboring a JAK defect (MUTZ5 cell line). Mice were euthanized at 5 weeks and BM was harvested. Evaluation of BM disease by flow cytometry showed that the percentage of viable human leukemia cells in hTSLP+ mice was twice that observed in hTSLP– mice. Evaluation of cell cycle progression in human CRLF2 B-ALL cells isolated from xenograft BM showed that the percentage of cycling cells in hTSLP+ mice was 2.5 fold higher than in hTSLP– mice. When primary Ph-like ALL cells were transplanted to produce hTSLP+/– xenografts, the viable pre-B ALL cells present in the BM of hTSLP+ mice showed higher expression levels of the TSLPR components (CRLF2 and IL-7Rα) than those in the hTSLP- mice. These data provide evidence that the TSLP produced in this model is active and that it impacts primary pre-B ALL cells. Preliminary data obtained from this model suggests that TSLP provides a signal that promotes in vivo survival of CRLF2 B-ALL cells and that it may play a role in selection of leukemia clones during in vivo leukemogenesis. Microarray analysis comparing gene expression in primary CRLF2 B-ALL cells isolated from hTSLP+ and hTSLP– xenograft mice identified 565 that genes are differentially regulated (&gt; 2 fold up or downregulated; p&lt;.05). Ingenuity Pathway Analysis is currently underway to identify the signaling pathways that are regulated by hTSLP in CRLF2 B-ALL in vivo in the hTSLP+/– xenograft model. The identification of genes downstream of TSLP-CRLF2 signaling has the potential of providing drug targets for combination therapy to effectively treat Ph-like B-ALL. The hTSLP+/– xenograft model will provide an important tool for evaluating the in vivo efficacy of these and other drugs to treat CRLF2 B-ALL.
Citation Format: Olivia Francis, Ruijun Su, Shannalee Martinez, Ineavely Baez, Terry-Ann Milford, Terrence Bennett, Ross Fisher, Christopher L. Morris, Sinisa Dovat, Kimberly J. Payne. A human-mouse xenograft model to evaluate therapies and study the role of TSLP-induced signals in Ph-like ALL. [abstract]. In: Proceedings of the AACR Special Conference on Pediatric Cancer at the Crossroads: Translating Discovery into Improved Outcomes; Nov 3-6, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;74(20 Suppl):Abstract nr B21.