T hymic stromal lymphopoietin (TSLP) stimulates in vitro proliferation of human fetal B-cell precursors. However, its in vivo role during normal human B lymphopoiesis is unknown. Genetic alterations that cause overexpression of its receptor component, cytokine receptor-like factor 2 (CRLF2), lead to high-risk B-cell acute lymphoblastic leukemia implicating this signaling pathway in leukemogenesis. We show that mouse thymic stromal lymphopoietin does not stimulate the downstream pathways (JAK/STAT5 and PI3K/AKT/mTOR) activated by the human cytokine in primary high-risk leukemia with overexpression of the receptor component. Thus, the utility of classic patient-derived xenografts for in vivo studies of this pathway is limited. We engineered xenograft mice to produce human thymic stromal lymphopoietin (+T mice) by injection with stromal cells transduced to express the cytokine. Control (-T) mice were produced using stroma transduced with control vector. Normal levels of human thymic stromal lymphopoietin were achieved in sera of +T mice, but were undetectable in -T mice. Patient-derived xenografts generated from +T as compared to -T mice showed a 3-6-fold increase in normal human B-cell precursors that was maintained through later stages of B-cell development. Gene expression profiles in high-risk B-cell acute lymphoblastic leukemia expanded in +T mice indicate increased mTOR pathway activation and are more similar to the original patient sample than those from -T mice. +T/-T xenografts provide a novel pre-clinical model for understanding this pathway in B lymphopoiesis and identifying treatments for high-risk B-cell acute lymphoblastic leukemia with overexpression of cytokine-like factor receptor 2.

Diamond Blackfan anemia (DBA), a syndrome primarily characterized by anemia and physical abnormalities, is one among a group of related inherited bone marrow failure syndromes (IBMFS) which share overlapping clinical features. Heterozygous mutations or single-copy deletions have been identified in 12 ribosomal protein genes in approximately 60% of DBA cases, with the genetic etiology unexplained in most remaining patients. Unlike many IBMFS, for which functional screening assays complement clinical and genetic findings, suspected DBA in the absence of typical alterations of the known genes must frequently be diagnosed after exclusion of other IBMFS. We report here a novel deletion in a child that presented such a diagnostic challenge and prompted development of a novel functional assay that can assist in the diagnosis of a significant fraction of patients with DBA. The ribosomal proteins affected in DBA are required for pre-rRNA processing, a process which can be interrogated to monitor steps in the maturation of 40S and 60S ribosomal subunits. In contrast to prior methods used to assess pre-rRNA processing, the assay reported here, based on capillary electrophoresis measurement of the maturation of rRNA in pre-60S ribosomal subunits, would be readily amenable to use in diagnostic laboratories. In addition to utility as a diagnostic tool, we applied this technique to gene discovery in DBA, resulting in the identification of RPL31 as a novel DBA gene.

3476 Most cases of Diamond Blackfan anemia are caused by haploinsufficiency for genes encoding proteins of the large or small ribosomal subunit. All of the ribosomal proteins affected in DBA are essential components of the ribosome required for the assembly of their respective subunits, including processing of the primary pre-rRNA transcript to mature 18S, 5.8S, and 28S rRNAs. Pre-rRNA processing signatures associated with ribosomal protein haploinsufficiency demonstrate a role for individual proteins in subunit assembly and can differ depending on which protein is affected. A facile pre-rRNA processing assay that can discriminate between loss of function alleles for different ribosomal protein genes would be an invaluable aide to DBA diagnosis and gene discovery efforts. Such an assay could also provide insight into different aspects of DBA pathophysiology. We have developed a robust procedure to assess pre-rRNA processing patterns in activated lymphocytes from the peripheral blood of patients with known or suspected Diamond Blackfan anemia. This assay typically involves the electrophoretic separation of total RNA from activated lymphocytes followed by Northern blotting with various hybridization probes to different rRNA precursors. Using this assay, we have found a common 32S pre-rRNA processing intermediate present in RNA from DBA patients with mutations in virtually all known large ribosomal subunit genes. This 32S pre-rRNA can be visualized in situ in gels stained with ethidium bromide (see accompanying figure) greatly simplifying the identification of large subunit ribosomal protein genes harboring loss of function mutations. As more and more ribosomal protein genes are identified within the DBA population, it has become increasingly important to distinguish between variants that affect ribosomal protein function and benign polymorphisms. Therefore, an analysis of pre-rRNA processing can be used to identify causative genes in patients with complex genotypes where sequence variants are found in more than one ribosomal protein gene. We analyzed a patient with variants in genes encoding RPS19 and RPL11, two known DBA genes, plus a deletion containing the RPL31 gene that has not been previously linked to DBA. Data analyses overwhelmingly support the deletion of RPL31 as the causative lesion in this patient and identify RPL31 as a new DBA gene. Analysis of pre-rRNA processing can also guide additional gene discovery efforts. We performed pre-rRNA processing studies on two patients lacking mutations in known DBA genes. In one case, we observed a clear defect in the18S rRNA pathway, implicating a gene involved in the biogenesis of the small ribosomal subunit, whereas in a second patient there is no evidence of a ribosome biogenesis defect suggesting that the underlying mutation may not affect ribosome synthesis. These results will help guide further efforts to identify causative genes in this patient cohort. Finally, we have used pre-rRNA processing patterns to begin to examine the mechanisms underlying remission in DBA patients. To date, we have examined two samples from DBA patients in remission and showed the ribosome synthesis defect is retained even while in remission for one of these patients, whereas the pre-rRNA processing defect has resolved in the other patient. Disclosures: No relevant conflicts of interest to declare.

A subset of children with B cell precursor acute lymphoblastic leukemia (B-ALL) are at high risk for relapse and death. Gene expression profiles in these high-risk B-ALLs is similar to that of Philadelphia chromosome-positive ALL. Approximately half of these Ph-like B-ALL are characterized by genetic defects resulting in overexpression of CRLF2. This defect occurs 5 times more commonly in Hispanic children than others and thus is a significant biological component of pediatric cancer health disparities. CRLF2, together with the IL-7Rα, forms a receptor complex that is activated by the cytokine, TSLP. Receptor complex activation leads to JAK-STAT5 phosphorylation. The activating JAK mutations found in some CRLF2 B-ALL led to speculation that TSLP stimulation is not a factor in CRLF B-ALL. However, we and others have found that TSLP increases STAT5 phosphorylation in CRLF2 B-ALL cells, including those with JAK defects. Our next step was to evaluate the role of TSLP-CRLF2 interactions in vivo in the human-mouse xenograft model. However, mouse TSLP 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 may contribute to high risk CRLF2 B-ALL. 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). Then we used this hTSLP+/- xenograft model system to evaluate the in vivo effects of TSLP on transplanted CRLF2 B-ALL cells harboring a JAK defect (MUTZ5 cell line). Transplanted mice were euthanized at 5 weeks and bone marrow (BM) was harvested. Evaluation of BM by flow cytometry showed that approximately half of the human leukemia cells were apoptotic in mice without TSLP while apoptosis was virtually absent in CRLF2 B-ALL cells harvested from hTSLP+ mice. Next we used Ingenuity Pathway Analysis to identify functions and pathways regulated by TSLP. Primary CRLF2 B-ALL cells were transplanted into hTSLP+ and hTSLP- mice. Whole genome microarray performed on primary human leukemia cells isolated from BM of xenograft mice identified 280 genes that were upregulated and 281 genes that were downregulated (> 2 fold up or downregulated; p<.05) in leukemia cells from hTSLP+ as compared to hTSLP- mice. Ingenuity Pathway Analysis of changes in gene expression identified “Cell Death and Survival” as the “Molecular and Cellular Function” most impacted by TSLP (34 genes differentially regulated).These data suggest that TSLP-induced CRLF2 signaling may contribute to leukemia cell survival in vivo in CRLF2 B-ALL. Current studies are aimed at identifying TSLP-regulated genes that can be therapeutically targeted as a part of combination therapy to successfully treat CRLF2 B-ALL and reduce the cancer health disparities for children with this disease. Citation Format: Olivia L. Francis, Ruijun Su, Shannalee R. Martinez, Ineavely Baez, Terry-Ann Milford, Ross Fisher, Christopher L. Morris, Xiaobing Zhang, Valeri Filippov, Sinisa Dovat, Kimberly J. Payne. TSLP regulates expression of genes involved in cell survival in a preclinical xenograft model of CRLF2 B-ALL. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3097. doi:10.1158/1538-7445.AM2014-3097

Hispanic children with Acute Lymphoblastic Leukemia (ALL) have a 39% higher death rate than white children. A major contributor to this disparity is the lack of therapies that specifically target CRLF2 B cell ALL. This high-risk leukemia occurs five times more frequently among children of Hispanic/Latino ethnicity than others and represents the most significant biological component of childhood cancer health disparities identified to date. CRLF2 B-ALL is caused by genetic alterations that result in B cells that overexpress the cytokine receptor component, CRLF2, leading to malignant transformation and high-risk leukemia with poor prognosis. CRLF2 pairs with the IL-7Rα, to form a receptor complex that is activated by the cytokine, TSLP. Activation of the receptor complex stimulates downstream JAK-STAT5 phosphorylation that has been shown to induce proliferation and survival of B-ALL cells. Activating JAK mutations are found in many cases of CRLF2 B-ALL and have led to speculation that stimulation of the receptor by TSLP is not a factor in this disease. In preliminary studies to address this question we evaluated the effect of TSLP in CRLF2 B-ALL cells with JAK defects. Our data show that TSLP increases STAT5 phosphorylation in these cell lines and also in primary CRLF2 B-ALL cells. These data suggest that TSLP can activate JAK-STAT5 signaling to induce downstream survival and proliferation in CRLF2-B-ALL cells, including those with JAK defects. Bone marrow (BM) provides the tumor microenvironment that can harbor chemoresistant B-ALL cells responsible for relapse in B-ALL. We used RT-PCR and ELISA assays to show that TSLP is expressed in BM cells from pediatric patients and thus could provide an in vivo source of TSLP to stimulate CRLF2 B-ALL cells. Human-mouse xenograft models produced by transplanting human leukemia cells into immune deficient mice mimic the in vivo environment and are the model of choice for identifying therapies that target the mechanisms of chemoresistance that are characteristic of high-risk leukemia. 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 contains CRLF2. Thus, traditional xenograft models do not provide the TSLP-CRLF2 interactions that our data implicate as a contributing factor in CRLF2 B-ALL. To overcome this obstacle we have engineered immune deficient mice to express human TSLP (hTSLP+ mice) as well as control mice that lack the TSLP cytokine (hTSLP– mice). ELISA assays show hTSLP levels in the hTSLP+ mice that approximate the normal range in human plasma. We used this hTSLP+/- xenograft model system to study the in vivo effects of TSLP on mice transplanted with a CRLF2 B-ALL cell line harboring a JAK defect (MUTZ5) and with primary pre-B ALL cells from a Hispanic patient. Mice were euthanized at 5 weeks and BM disease was evaluated. In recipients of MUTZ5 B-ALL cells the percentage of viable leukemia cells in hTSLP+ mice was twice that observed in hTSLP- mice. Similarly, in recipients of primary B-ALL, the percentage of viable leukemia cells was higher in hTSLP+ than hTSLP- mice. These data provide evidence that the TSLP produced in this model is active and that TSLP-CRLF2 interactions contribute in vivo to CRLF2-B-ALL. This model will be particularly important for identifying therapies that can effectively target CRLF2 B-ALL and reduce cancer health disparities in Hispanic childhood B-ALL. Citation Format: Francis L. Olivia, Shannalee R. Martinez, Terrence Bennett, Ineavely Baez, Terry-Ann Milford, Christopher L. Morris, Ross O. Fisher, Xiao-Bing Zhang, Rui-Jun Su, Sinisa Dovat, Kimberly J. Payne. A novel preclinical model to identify therapies for CRLF2 B-ALL and reduce childhood cancer health disparities. [abstract]. In: Proceedings of the Fifth AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2012 Oct 27-30; San Diego, CA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2012;21(10 Suppl):Abstract nr B32.