Abstract A07: A novel patient-derived xenograft model to define the role of TSLP-induced CRLF2 signals and identify therapies for Ph-like B-ALL
Authors: Francis Olivia L. , Milford Terry-Ann , Baez Ineavely , Coats Jacqueline S. , Morris Christopher L. , Fisher Ross , Handel Ben Van , Su Ruijun , Suterwala Batul , Kamal Muhammad , Gohar Shadi Farzin , Dovat Sinisa , Payne Kimberly J. 
Published: May 2016

A subset of high-risk B cell acute lymphoblastic leukemia (ALL) shows a gene expression profile similar to Philadelphia chromosome positive (Ph+) ALL and has been described as Ph-like ALL. Approximately 50% of 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. 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. While activating JAK mutations are associated with CRLF2 B-ALL, over half of CRLF2 B-ALL lack such mutations. Our data show that primary human bone marrow (BM) stromal cells express TSLP. Thus TSLP is present in the tumor microenvironment to provide TSLP-induced CRLF2 signals that could play a role in the initiation, maintenance and/or progression of CRLF2 B-ALL. 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%) with respect to human TSLP and CRLF2. Further, phospho flow cytometry assays show that human, but not mouse TSLP activates CRLF2 signals in primary human CRLF2 B-ALL cells and cell lines as indicated by increased pSTAT5, pAKT and pS6. These data indicate that the mouse TSLP present in classic patient derived xenograft models (PDX) does not produce the TSLP-induced CRLF2 signals present in the patient. To address this challenge we engineered 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 transplantation 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 human CRLF2 B-ALL cells. ELISA assays showed that serum levels of hTSLP in mice was proportional to numbers of stromal cells injected at weekly time points. Normal human serum levels of hTSLP (12-32 pg/ml) could be achieved in +T mice, while hTSLP was undetectable in T mice. Because 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 CRLF2 B-ALL leukemia cells. Human CRLF2 B-ALL cells were isolated from the BM of PDX mice and whole genome microarray was performed. 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. To determine whether +T PDX mice provide a preclinical model of B-ALL that more closely mirrors patients than T PDX mice, we compared RNAseq gene expression profiles of leukemia cells from +T and T PDX mice to that from original patient sample. The gene expression pattern in +T mice was significantly closer to primary patient sample than that 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.

Citation Format: Olivia L. Francis, Terry-Ann Milford, Ineavely Baez, Jacqueline S. Coats, Christopher L. Morris, Ross Fisher, Ben Van Handel, Ruijun Su, Batul Suterwala, Muhammad Kamal, Shadi Farzin Gohar, Sinisa Dovat, Kimberly J. Payne. A novel patient-derived xenograft model to define the role of TSLP-induced CRLF2 signals and identify therapies for Ph-like B-ALL. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Pediatric Cancer Research: From Mechanisms and Models to Treatment and Survivorship; 2015 Nov 9-12; Fort Lauderdale, FL. Philadelphia (PA): AACR; Cancer Res 2016;76(5 Suppl):Abstract nr A07.