A Patient-Derived Xenograft Model for Identifying Therapies and Defining Mechanisms of TSLP-Induced CRLF2 Signals in Ph-like B-ALL
Authors: Francis Olivia L , MIlford Terry-Ann , Baez Ineavely , Coats Jacqueline , Morris Christopher L. , Fisher Ross , Van Handel Benjamin Joseph , Su Ruijun , Suterwala Batul T. , Farzingohar Shadi , Dovat Sinisa , Payne Kimberly 
Published: October 2019

Abstract
Philadelphia chromosome (Ph)-like B cell acute lymphoblastic leukemia (B-ALL) is a high-risk leukemia with a gene expression profile similar to BCR-ABL1+ B-ALL. Approximately 50% of all Ph-like B-ALL is characterized by genetic alterations leading to overexpression of CRLF2 (CRLF2 B-ALL). CRLF2 B-ALL occurs 5 times more often in Hispanic and Native American children than others and is prevalent in adolescents and young adults. The poor outcomes associated with CRLF2 B-ALL represent a major clinical challenge and an important component of pediatric cancer health disparities. Biologically, CRLF2 acts as a receptor component for the cytokine, TSLP, which induces JAK2-STAT5 and PI3/AKT/mTOR pathway activation downstream of binding to CRLF2. Activating JAK mutations are associated with CRLF2 B-ALL, but overall data indicate that JAK mutations are present in 50% or less of CRLF2 B-ALL. Our data show that normal primary human bone marrow (BM) stromal cells express TSLP, suggesting that TSLP-induced CRLF2 signals could play a role in the initiation, maintenance and progression of CRLF2 B-ALL, particularly in cases without JAK mutations. Consistent with this, TSLP has been reported to increase in vitro production of human fetal B cell precursors. However studies of TSLP in B lymphopoiesis have been conducted almost exclusively in mice which show low homology (~40%) to human TSLP and CRLF2. Further, using phospho flow cytometry we show that mouse TSLP is unable to induce increases in pSTAT5, pAKT and pS6 observed in CRLF2 B-ALL cells stimulated with human TSLP, confirming the species specificity of mouse TSLP. These findings underscore the importance and challenge of developing in vivo systems that can model human TSLP-CRLF2 interactions for evaluating therapies and studying leukemogenesis of CRRLF2 B-ALL. To address this challenge we engineered patient-derived xenograft (PDX) mice to produce human TSLP (hTSLP) by transplanting them with stromal cells transduced to express hTSLP (+T mice). Control (-T) mice were produced by transplanting with stroma transduced with a control vector. Supernatant from engineered +T stroma, but not -T stroma, induced JAK/STAT5 and PI3K/AKT/mTOR pathway activation in CRLF2 B-ALL cells. ELISA assays showed normal serum levels of hTSLP (12-32 pg/ml) in +T mice, while hTSLP was undetectable in -T mice. Since TSLP has been shown to increase in vitro production of human B cell precursors, we evaluated the in vivo functionality of our model by comparing the production of normal B cell precursors in the BM of +T and -T PDX mice generated with human umbilical cord blood CD34+ cells. Data from 3 different cord blood donors showed that production of B cell precursors is 3-5 fold increased in +T as compared to -T mice. TSLP-induced increases were specific to B lineage cells, initiated in the earliest CD19+ B cell precursors, and maintained through later stages of B cell development. Next we evaluate the in vivo functionality of our model using primary leukemia cells. +T and -T PDX mice were produced using primary CRLF2 B-ALL cells. BM was harvested and whole genome microarray was performed on isolated CRLF2 B-ALL cells. Evaluation of microarray data by Gene Set Enrichment Analysis (GSEA) and Ingenuity Pathway Analysis showed that genes downstream of mTOR pathway activation were upregulated in +T as compared to -T PDX mice, confirming hTSLP activity in the +T PDX mice. Next we tested whether +T PDX mice provide an in vivo model of B-ALL that more closely mirrors patients than -T PDX mice. +T and -T PDX mice were generated from primary high risk B-ALL. RNAseq gene expression profiles from primary patient B-ALL cells were compared to those of the same patient sample expanded in +T and -T PDX mice. The gene expression pattern in +T mice was significantly closer to the primary patient sample than those from -T mice. The +T and -T PDX mice described here provide a novel preclinical model for studying the role of TSLP in the initiation, progression and maintenance of CRLF2 B-ALL and for evaluating drug efficacy in an in vivo model that more closely mirrors the in vivo environment present in patients.

Disclosures
No relevant conflicts of interest to declare.