The Novel Technique of Genetically Engineered Patient-Derived Xenografts (GEPDX) Reveals That the X-Linked Inhibitor of Apoptosis Protein (XIAP) Plays an Essential Role for Maintenance and Growth of Patients' Acute Lymphoblastic Leukemia In Vivo

Expression profiling and next generation sequencing have enabled a detailed knowledge on alterations present in tumors from individual patients. In contrast, only limited understanding exists on the role that each alteration plays for the existing tumor. Of direct clinical interest, genes are of special interest which harbor an essential function for tumor maintenance and growth as they represent putative targets for anti-cancer therapy.

Characterizing gene functions is demanding regarding both techniques and resources. Questions on gene function are often studied in established tumor cell lines, although establishing cell lines from primary tumors is rarely successful in acute leukemias. Primary acute leukemia cells poorly grow in vitro and established acute leukemias cell lines rely on additional mutations enabling in vitro growth, making them doubtful models to study genes with essential function.

To bridge the gap, we aimed at studying gene function in the complex environment of individual tumors. As primary acute leukemia cells are unable to grow in vitro, we used the orthotopic model of patient-derived xenograft (PDX) leukemia and amplified cells in mice.

We established a novel technique to manipulate distinct signaling proteins in PDX cells using lentiviruses and knockdown. We expressed small hairpin RNA (shRNA) in the background of micro RNA 30 (miR30) under control of a Pol II promoter and 3 prime of dsRED as molecular marker. This approach closely links expression of the shRNA to the fluorochrome and resulted in a potent and stable knockdown. We expressed a control shRNA targeting Renilla luciferase and several shRNA sequences targeting XIAP. In order to discriminate different derivative cell populations within a single mouse, we co-expressed a second fluorochrome from a second plasmid so that green cells harbored a knockdown of XIAP, while blue cells harbored the control construct, thus allowing in vivo outcompete proliferation assays. We called our new approach "genetically engineered PDX (GEPDX)" models in parallel to genetically engineered mouse models (GEMM).

We used our new technology to study the role of XIAP, the X-linked inhibitor of apoptosis for acute lymphoblastic leukemia (ALL), the single most frequent tumor in children. XIAP is frequently and highly overexpressed in hematological malignancies and its up-regulation was shown to be associated with inferior prognosis of patients in different tumors. Nevertheless XIAP's role for tumor maintenance remains unclear.

In several preB- and T-cell ALL cell lines, potent and stable knockdown of XIAP did not alter cell proliferation in vitro or upon xeno-transplantation in vivo. Thus expression levels of XIAP seem irrelevant for spontaneous proliferation of established acute leukemia cell lines in vitro and in vivo.

We next studied PDX ALL cells growing in mice as model closer related to patients. We generated GEPDX cells from two children with relapse of a B precursor ALL with either knockdown of XIAP or control knockdown together with the appropriate molecular color markers. When blue and green GEPDX cells harboring control or XIAP knockdown, respectively, were co-transplanted into mice at a 1:1 ratio in a competitive outcompete assay, control transfected cells significantly overgrew or even eliminated cells with XIAP knockdown in both samples studied. GEPDX cells with knockdown of XIAP showed a significant and dose-dependent growth disadvantage in vivo compared to control cells indicating that XIAP played an essential role for PDX cells growing in vivo.

Thus, our novel technique of genetic engineering in PDX cells revealed an essential role of XIAP for tumor maintenance and growth in patients' tumor cells making XIAP an attractive therapeutic target in ALL. As established ALL cell lines were unable to unravel this important role of XIAP, GEPDX might be superior to cell lines for identifying genes with essential function. GEPDX represent a powerful new tool to characterize the complex environment of individual patients' tumor cells in vivo, the function of the many lesions and alterations described by expression profiling and next generation sequencing.