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Human urinary disorders are generally studied in rodent models due to limitations of functional in vitro culture models of primary human urothelial cells (HUCs). Current HUC culture models are often derived from immortalized cancer cell lines, which likely have functional characteristics differ from healthy human urothelium. Here, we described a simple explant culture technique to generate HUCs and assessed their in vitro functions. Using transmission electron microscopy, we assessed morphology and heterogeneity of the generated HUCs and characterized their intercellular membrane structural proteins relative to ex vivo urothelium tissue. We demonstrated that our cultured HUCs are free of fibroblasts. They are also heterogeneous, containing cells characteristic of both immature basal cells and mature superficial urothelial cells. The cultured HUCs expressed muscarinic receptors (MR1 and MR2), carnitine acetyltransferase (CarAT), immunoregulatory cytokines IL7, IL15, and IL23, as well as the chemokine CCL20. HUCs also expressed epithelial cell-specific molecules essential for forming intercellular structures that maintain the functional capacity to form the physiological barrier of the human bladder urothelium. A subset of HUCs, identified by the high expression of CD44, expressed the Toll-like receptor 4 (TLR4) along with its co-receptor CD14. We demonstrated that HUCs express, at the mRNA level, both forms of the IL22 receptor, the membrane-associated (IL22RA1) and the secreted soluble (IL22RA2) forms; in turn, IL22 inhibited expression of MR1 and induced expression of CarAT and two antimicrobial peptides (S100A9 and lipocalin-2). While the cellular sources of IL22 have yet to be identified, the HUC cytokine and chemokine profiles support the concept that IL22-producing cells are present in the human bladder mucosa tissue and that IL22 plays a regulatory role in HUC functions. Thus, the described explant technique is clearly capable of generating functional HUCs suitable for the study of human urinary tract disorders, including interactions between urothelium and IL22-producing cells.

Mixed lineage leukemias are characterized by the creation of a chimeric fusion protein where MLL is fused in frame to over 60 different fusion partners, leading to the disruption of HOX gene regulation. AF9 is one of the most common MLL fusion partners, and MLL-AF9 leukemia is acute and aggressive with a poor overall prognosis. The mechanism by which AF9 regulates normal transcription and contributes to dysregulated transcription is poorly understood. We have shown that the C-terminal domain of AF9 binds to four different proteins, two of which (Dot1L – an H3K79 methyltransferase and AF4 – which recruits P-TEFb), are transcriptional activators, whereas the other two (CBX8 – which is a part of the PRC1 repressive complex and BCOR- BCL6 corepressor), are generally transcriptional repressors, suggesting that AF9 acts as a protein signaling hub. We have previously shown that the C-terminal domain of AF9 is an intrinsically disordered protein (IDP) meaning that it is unstructured on its own but undergoes coupled folding and binding upon interacting with its binding partners1. Our goal is to use structure-function studies to further understand AF9 protein interactions and to elucidate which of these interactions with AF9 are critical for MLL-AF9 leukemogenesis. We have determined NMR solution structures of complexes of Dot1L and CBX8 with AF9. Both Dot1L and CBX8 complexes form mixed alpha-beta structures, similar to that of our previously solved AF4-AF9 complex structure where AF4, Dot1L, and CBX8 all have a consensus LXVXIXL sequence and form a ß-strand and an extended ß-turn. Intriguingly, Dot1L contains three separate binding motifs for AF9 interaction, including one repeat motif separated by seven amino acids. Our 15N-1H HSQC NMR experiments show that each of these three binding sites on Dot1L adopts a similar fold with AF9 yet has vastly different binding affinities with AF9. We have created several structure-guided mutations on AF9 that differentially block interactions of its binding partners. A single charge reversal mutation on AF9 that blocks all protein interactions with AF9 was introduced into MLL-AF9 in vitro colony forming assays, and was able to abrogate the serial replating capacity of MLL-AF9. Other mutations that selectively block the interaction of various proteins with AF9, show differential effects in colony forming assays and HOX gene expression suggesting the importance of certain AF9 protein interactions in MLL-AF9 leukemogenesis. 1. Leach, B. I. et al. Leukemia fusion target AF9 is an intrinsically disordered transcriptional regulator that recruits multiple partners via coupled folding and binding. Structure21, 176-183, doi:10.1016/j.str.2012.11.011 (2013). Disclosures: No relevant conflicts of interest to declare.

Foxn1 is essential for thymic organogenesis and T lymphopoiesis. While reduced Foxn1 expression results in a decline in T lymphopoiesis, overexpression of Foxn1 in the thymus of a transgenic mouse model (Foxn1Tg) attenuates the age-associated decline in T lymphopoiesis. T lymphopoiesis begins with ETP, derived from MPP in the BM. A decline in MPP and ETP numbers with age is thought to contribute to reduced T lymphopoiesis. Previously, we showed that reduced ETP number with age is attenuated in Foxn1Tg; whether the effect is initiated in the BM with MPP is not known. Here, we report that Foxn1 is expressed in Wt BM and over expressed in Foxn1Tg. With age, the number of MPP in Foxn1Tg was not reduced, and Foxn1Tg also have a larger pool of HSC. Furthermore, the Foxn1Tg BM is more efficient in generating MPP. In contrast to MPP, CLP and B lineage cell numbers were significantly lower in both young and aged Foxn1Tg compared to Wt. We identified a novel population of lineageneg/low, EpCAMpos, CD138neg cells as Foxn1-expressing BM cells that also express Delta-like 4. Thus, Foxn1 affects both T lymphopoiesis and hematopoiesis, suggesting that the Foxn1 BM niches function in skewing MPP development toward T lineage progenitors.

Abstract 1878 NF-κB activation is essential for leukemic cell and stem cell (LSC) survival and self-renewal, but is significantly less essential for similar functions in normal bone marrow hematopoietic stem/progenitor cells (HSPCs). As a result, LSCs are more sensitive to both pharmacologic and genetic NF-κB inhibition than HSPCs. These sensitivities suggest that NF-κB signaling could be a potential therapeutic target in the treatment of leukemia. However, high doses of NF-κB inhibitor treatment are also associated with significant inflammation-mediated toxicity to liver, skin and other tissues. Therefore, new approaches are needed that will be able to protect normal tissues while simultaneously enhancing the effects of NF-κB inhibition on leukemic cells. By utilizing genetic knock-out HSPC/leukemia models in combination with small molecule inhibitors, we searched for factors that could sensitize leukemic cells to NF-κB inhibition while simultaneously protecting HSPCs. We demonstrated that targeted inhibition of TNFα induced NF-κB-independent signaling would be a useful approach to treat leukemia in combination with NF-κB inhibition. We found that deactivating TNFα signaling either by genetic deletion of its receptors or through neutralizing the ligand with an antibody can significantly enhance NF-κB inhibition-induced leukemic cell elimination. In contrast, deactivation of TNFα signaling can significantly protect normal HSPCs from NF-κB inhibitor-induced death. Mechanistic studies revealed that TNFα stimulates several similar signals in both leukemic cells and HSPCs, including NF-κB, ERK, AKT, p38 and JNK. In order to determine which of these signals would best augment NF-κB inhibition, we performed biochemical analyses and searched for candidate survival signals activated downstream of TNFα and that operated independently of NF-κB. This analysis revealed that TNFα-induced ERK and AKT signals are NF-κB dependent, while TNFα-induced p38 and JNK signals are NF-κB independent. Inhibition of p38 enhanced leukemic cell growth, and was therefore ruled out as a candidate. Our analyses showed that JNK was activated by TNFα stimulation, operated independently of NF-κB activation, and also repressed leukemic cell growth. Further study confirmed that TNFα-dependent JNK activation has opposite functions in HSPCs and leukemic cells: JNK acts by promoting cell survival in leukemic cells while inducing cell death in HSPCs. We confirmed this result by inactivating the JNK signal via small molecule inhibitor, and found that we could significantly sensitize leukemic cells to NF-κB inhibition while protecting normal HSPCs from TNFα-mediated cell death associated with NF-κB inhibition. Mechanism analysis suggested that TNFα represses the growth of HSPCs by a JNK-RIP1/RIP3-dependent necroptosis mechanism, whereas TNFα promotes the expansion of leukemic cells by inducing the parallel activation of NF-κB-dependent AKT/ERK signaling and NF-κB-independent JNK signaling. In conclusion, our studies suggest the simultaneous inhibition of both NF-κB and TNFα-induced NF-κB-independent signals like JNK might provide a more comprehensive approach for targeted treatment of leukemias that also protects against deleterious inflammation in the bone marrow and other tissues. Disclosures: Nand: Celgene: Research Funding.

The thymus requires continuous seeding of progenitors from hematopoietic stem cells (HSC) in bone marrow (BM) for naïve T cell production. HSC give rise to multipotent progenitors (MPP) and their progeny common lymphoid progenitors (CLP); however, MPP are the predominant source of early T cell progenitors (ETP) in the thymus. ETP, HSC, MPP and CLP decline with age, culminating in reduced naïve T cell production. We reported that the age-associated decline in ETP is abrogated in K14-Foxn1 transgenic mice (Foxn1Tg). Here, we show that Foxn1 is expressed in BM and its expression was 98-fold higher in Foxn1Tg. Foxn1 transgene expression increased endogenous Foxn1 mRNA levels by 18- to 24-fold; Foxn1 positive cells were readily detected in the BM. We found that HSC number was 2-fold higher in young Foxn1Tg versus wild type (Wt). HSC and MPP numbers declined in aged Wt but not in Foxn1Tg. A higher percent of HSC was in S,G2/M; further, HSC and MPP in subGo from Foxn1Tg were respectively 3-fold and 4-fold reduced, suggesting inhibition of cell death. In contrast, CLP frequency was 4-fold less in young Foxn1Tg. A higher number of the committed T cell progenitors (CTP) with a 2-fold increase in S,G2/M fraction were found in young Foxn1Tg versus Wt. Our data provide evidence linking Foxn1 to the development of T cell progenitors in the BM and thymus, suggesting that increased expression could limit the decline in naïve T cell production with age.

Abstract 1557 In bone marrow (BM) hematopoietic stem cells/progenitors (HSC/Ps), the apoptotic machinery is tightly controlled by a complex interplay between intrinsic signals and stimuli from the surrounding microenvironment, inducing a dynamically balanced network between pro-apoptotic and anti-apoptotic influences. Disruption of this balance can result in hematopoietic disorders such as various BM failure syndromes. Studies have suggested that tumor necrosis factor-α (TNF-α) and Fas-ligand induce programmed cell death and/or differentiation of HSC/Ps, thus exercising negative regulation over hematopoiesis. However, whether the apoptotic cell death induced by these factors plays a role in BM failure syndromes remains ambiguous. We have reported that transforming growth factor beta-activated kinase-1 (TAK-1) plays an essential role in the survival of HSC/Ps. Mice with TAK-1 deletion in BM hematopoietic cells develop BM failure due to the apoptotic death of mutant HSC/Ps. We have taken advantage of the dramatic phenotypes of this mutant mouse line to closely examine TNF-α and Fas signaling in order to understand which of these is related to the induction of apoptosis of HSC/Ps, and to what degree. To do so, we generated TAK-1/TNFR1a, TAK-1/TNFR1b and TAK-1/Fas compound-mutant mice in order to evaluate which signaling system, when inactivated, permitted the rescue the BM failure defects observed in TAK-1 knockout mice, and the degree to which it did so, using both in vivo and in vitro studies. We found that, as was the case with TAK-1 knockout mice, TAK-1/TNFR1b and TAK-1/Fas compound-mutant mice died within 8 to 10 days after induction of TAK-1 deletion with exactly the same type of BM failure observed in TAK-1 knockout mice. In vitro studies indicated that neither TNFR1b nor Fas deletion was able to protect cells from apoptosis, nor could they rescue the colony-forming ability of TAK-1 mutant HSC/Ps. However, TAK-1/TNFR1a-mutant mice appeared to be healthy one month after induction of TAK1 deletion. By careful analysis, we found that TNFR1a deletion partially rescued the BM failure phenotype of TAK-1 knockout mice. The total numbers of nucleated BM and splenic cells in TAK-1/TNFR1a- mutant mice are approximately 54.7% and 83.8% (respectively) of those of their wild-type littermate controls. These percentages represent significant increases comparing to their littermates with TAK-1 deletion only (7.5% and 17% of WT control). In vitro studies demonstrated that TNFR1a deletion restored colony-forming ability in 20–30% of TAK1-knockout HSC/Ps. Currently, we are in the process of analyzing the hematopoietic phenotypes of TAK-1/TNFR1a/1b triple-mutant mice in order to determine whether the complete inactivation of TNF-α signaling further reverses the hematopoietic defects seen in TAK1-knockout mice. Our study demonstrated that TNF-α, via its receptor 1a-induced apoptosis, contributes substantially to the loss of HSC/Ps in TAK-1 knockout mice. Our results also suggest that pro-apoptotic signaling other than TNF-α is also involved in the BM failure syndrome observed in TAK-1 mutant mice. Disclosures: No relevant conflicts of interest to declare.

Abstract 1632 Poster Board I-658 c-Myc, the product of an oncogene, is a common target of most leukemic oncoproteins. Deregulation of c-Myc is commonly found in human leukemic blasts. Transgenic over-expression of c-Myc induces myeloid, erythroid, and lymphocytic leukemia in mice, however whether c-Myc is absolutely required for leukemogenesis has not yet been addressed. Pten, a tumor-suppressing phosphatase, inhibits cell proliferation and promotes apoptotic cell death through repression of PI3K-Akt signaling. Inactivating mutations of Pten and deregulation of PI3K/Akt signaling are both involved in the development of both chronic and acute hematopoietic malignances. Mice with Pten deletions in hematopoietic stem cells (HSCs) develop myeloproliferative disorders (MPD) followed by acute T lymphocytic or myeloid leukemia, reminiscent of disease progression in human chronic myelogenous leukemia. Mice with Pten deletions in lymphocytes develop lymphadenopathy (due to a chronic polyclonal lymphoproliferative disorder) as well as CD4+ T lymphocytic lymphoma. The appearance of these diseases in one animal model provides an opportunity to study the pathogenesis of multiple diseases simultaneously. To study whether c-Myc is required for the development of these hematopoietic disorders in Pten-mutant mice, we generated inducible Pten and c-Myc double-knockout mice (Pten-/-c-Myc-/-). By comparing the hematopoietic phenotypes of the Pten-/-c-Myc-/- mice with those of Pten-mutant (Pten-/-) mice, we found that both sets of mice developed MPD and lymphadenopathy, however none of the compound-mutant mice developed acute leukemia or lymphoma. Interestingly, in contrast to the MPD which developed in Pten-/- mice, which is dominated by granulocytes, megakaryocytes predominate in the MPD of Pten-/-c-Myc-/- mice. We have concluded that c-Myc is required for the development of both T lymphocytic lymphoma and the acute leukemic transformation of Pten-/- MPD, but is not essential for the development of chronic myeloid or lymphoid proliferative disorders. Our study suggests that deregulation of PI3K/Akt signaling in Pten-mutant hematopoietic cells protects these cells from apoptotic cell death, which results in chronic proliferative disorders, while the deregulation of c-Myc resulting from additional mutations promotes hematopoietic cell proliferation and blockage of maturation, and is absolutely required for the development of acute hematopoietic malignances. Disclosures No relevant conflicts of interest to declare.

The thymus is the primary site for thymopoiesis and production of naïve T cells; however, thymic function declines with age. Thymopoiesis requires continuous thymic seeding by early T cell progenitors (ETP), a progeny of the bone marrow-derived lineage marker negative, Sca1pos, c-kitpos (LSK) progenitors that express Flt3 (LSK Flt3pos). A decrease in the frequency of ETP, and in the number of LSK Flt3pos with age has been implicated as a mechanism for the decline in thymopoiesis. We have shown that a decrease in thymocyte numbers occurs as early as 3m of age and correlates with a decrease in Foxn1 expression; however, a role for Foxn1 in thymopoiesis remains unknown. Here, we show that thymi of aged Foxn1 transgenic mice (Tg) have a higher number of thymocytes and ETP compared to aged wild type mice. Forced expression of Foxn1 also rescues aged-associated alterations of thymic architecture. Unexpectedly, we found that Foxn1 is over expressed in the bone marrow (BM). While the LSK and LSK Flt3pos are reduced in aged wild type mice, these populations remain unchanged in aged Foxn1Tg. Furthermore, the aged Foxn1 Tg also have a higher number of the lineage marker negative CD90.2pos CD2pos, a population absent in the Foxn1 mutated nude mice. Thus, our data suggest that expression of Foxn1 is critical for functional thymic and BM stromal cell niches required for the survival of T cell progenitors (NIA AG023809).

The discovery of Delta-like-1 (Dl1)/Notch signaling as the earliest requirement for T lineage commitment and development makes it possible to generate T cells from hematopoietic stem cells in vitro. The mouse bone marrow stroma OP9 cells that overexpress Dl1is the first stroma cell line that is capable of promoting the development of both mouse and human T cells; however, a similar approach with human stroma cells has not been established. To develop a similar culture system to closely emulate human T cell development, we have generated a human TEC line from a pediatric thymus and expressed the mouse Dl1 in this line (TEC84-Dl1). The TEC84-Dl1 expresses CD104, CD29, CD49f, CD54 (ICAM-1), and CD106 (VCAM-1); these cells also express HLA-ABC and HLA-DR. When TEC84-Dl1 were co-cultured with cord blood CD34posCD45RAposCD38neg cells, we were able to generate preT/NK (CD34posCD7posCD1aneg), preT (CD34posCD7posCD1apos), immature single positive and double positive subsets; the co-culture system also generated a CD3pos subset. Similar results were also obtained with CD34pos cells isolated from adult bone marrow. Thus, our co-culture system with the human TEC provides a complete human system for replicating thymopoiesis in vitro and offers a novel tool to optimize the development of T lineage cells that would benefit cord blood and bone marrow transplant recipients (Supports: AG023809 to PL, IDPH to PS, AI068390 to KK).

Successful cord blood (CB) transplantation in adults is hampered by low numbers of hematopoietic stem cells (HSC) and infection due to delayed establishment of immunocompetence. By performing CDR3 spectratype analysis of T and B lymphocytes from CB transplant recipients, we demonstrated that these patients do not develop a diverse repertoire until 6 months or more after transplant. To provide rapidly engrafting antigen-responsive cells to CB recipients and to increase the number of transplanted cells, we developed an in vitro system to expand lymphoid progenitors from CB HSC. CB HSC (CD34+CD45RA+CD38-) are co-cultured for one to two weeks with either HEK 293T cells or a non-tumorigenic human thymic epithelial cell line (TEC) in media supplemented with a cocktail of cytokines and growth factors. In this study, we focused on B-lineage cells (CD79a+) and found, on average, B-lineage cells expanded more than 70 fold in HEK 293T co-cultures and nearly 100 fold in TEC co-cultures. We conclude that B-lineage cells can be expanded effectively in vitro; consequently, we are testing whether these cells are functional after engraftment in NOD/SCID/IL2Rγ-/- mice. We hypothesize that compared to patients receiving HSC alone, patients receiving transplants supplemented with in vitro-generated lymphoid progenitors will become immunocompetent sooner. Funding by NIH AI068390 (KLK) and AG023809 (PTL)

It was shown that c-Myc is required for the proliferation and differentiation of hematopoietic stem cells and progenitors. Mice with c-Myc deletions develop severe anemia and cytopenia. However, hematopoietic stem cells (HSCs) accumulate in significant numbers in the bone marrow (BM) of mutant mice, probably due to an increase in adhesiveness of the mutant HSCs to BM osteoblastic niche cells. Previously, we demonstrated that PTEN might play a critical role in the process of adhesion of HSCs to BM niche cells. Deletion of PTEN results in the proliferation and peripheral mobilization of HSCs, followed by a decline in these cells. PTEN mutant mice proceed to develop myeloproliferative disorders. Here we report that c-Myc also has an essential role to play in regulating the lineage commitment of HSCs and progenitors (HSC/Ps). HSC/Ps in which c-Myc is deleted are biased toward megakaryocytic lineage differentiation at the expense of other lineages. c-Myc knockout mice develop significant thrombocytosis (3- to 5-fold increase in peripheral platelet numbers) due to an obviously increased megakaryopoiesis in BM and spleen. PTEN deletion causes down-regulation of expression of adhesive molecules, including integrins and N-cadherin, in HSCs, resulting not only in an increased mobilization of c-Myc-mutant HSCs from the BM niche but also mobilization of c-Myc-mutant megakaryocytic progenitors to the spleen. We found that HSCs and megakaryocytic progenitors are significantly reduced in BM but dramatically increased in the spleens of PTEN/c-MYC double-knockout mice, compared to c-Myc single-gene knockout mice. In addition, PTEN deletion further promotes megakaryocytic progenitor cell proliferation, as well as infiltration of these cells into the liver. PTEN/c-Myc double-gene knockout mice consistently develop megakaryocytic proliferative disorders. We conclude that the ability of c-Myc to regulate HSC-BM niche interactions is at least partially accomplished through inhibition of PTEN function by c-Myc. In addition, c-Myc controls the lineage commitment of HSC/Ps. Deletion of c-Myc converts the myeloproliferative disorder seen in PTEN knockout mice to a megakaryocytic proliferative disorder. Whether PTEN and c-Myc mutations are likewise etiologically involved in human megakaryocytic proliferative disorders and megakaryocytic leukemia is currently a topic of active investigation in our laboratory.

TGFβ1-activated kinase 1 (TAK1), a member of the MAPKKK family, is a key mediator of stress and proinflammatory signals. TAK1 can be activated by inflammation-mediating cytokines, including tumor necrosis factor-α (TNF-α and interleukin-1b (IL-1β), as well as by T- and B- cell receptors (TCR/BCR), and Toll-like receptors (TLRs) signals. Activated TAK1 induces the nuclear localization of NF-kB and the activation of JNK/AP1 by stimulating IKKβ and MKK3/MKK6 phosphorylation respectively. TAK1 has been found to play an important role in inflammation, immunity, T- and B-cell activation, and epithelial cell survival. The TAK1−/ − phenotype is lethal in mice at the early embryonic stage. We found higher levels of TAK1 expression and activity in hematopoietic stem cells and progenitors (HSC/Ps), and reduced expression and activity in differentiated mature hematopoietic cells. To study the role of TAK1 in bone marrow hematopoiesis, we generated inducible-TAK1 knockout mice by crossing TAK1loxp mice with Mx1Cre mice, the latter being an interferon-inducible Cre mouse line. After injection of polyI:C to induce the knockout, we found that all the TAK1 knockout mice died within 8 to 10 days after the first polyI:C injection, showing severe hematopoietic and other defects; heterozygotes were phenotypically comparable to wild-type control animals. The TAK1 deletion in these mice resulted in ablation of bone marrow hematopoiesis due to the loss of C-Kit+ HSC/Ps. Annexin-V staining showed a 3-fold increase in apoptosis in the C-Kit+ HSC/Ps from TAK1 mutant mice compared to those from littermate control mice. Almost all of the mutant animals showed intestinal bleeding as well as other hemorrhaging due to the significant reductions in platelet counts. In reciprocal bone marrow transplantation experiments, we found that the TAK1-mutant bone marrow microenvironment was able to support the growth and function of wild-type HSC/Ps, while HSC/Ps from TAK1−/ − mice failed to grow within the wild-type bone marrow microenvironment. These observations suggest that the bone marrow ablation phenotype which develops in TAK1-mutant mice is the result of intrinsic defects in HSC/P’s. We propose that TAK1-mutant HSC/Ps might mediate a survival signal for HSC/Ps stimulated by hematopoietic growth factors and cytokines, such as stem cell factor (SCF). The details of possible mechanisms by which this phenomenon might occur is currently under active investigation by our group.

Dentin matrix protein 1 (DMP1) is expressed in both pulp and odontoblast cells and deletion of the Dmp1 gene leads to defects in odontogenesis and mineralization. The goals of this study were to examine how DMP1 controls dentin mineralization and odontogenesis in vivo. Fluorochrome labeling of dentin in Dmp1-null mice showed a diffuse labeling pattern with a three-fold reduction in dentin appositional rate compared to controls. Deletion of DMP1 was also associated with abnormalities in the dentinal tubule system and delayed formation of the third molar. Unlike the mineralization defect in Vitamin D receptor null mice, the mineralization defect in Dmp1-null mice was not rescued by a high calcium and phosphate diet, suggesting that the effects of DMP1 on mineralization are locally mediated. Re-expression of Dmp1 in early and late odontoblasts under control of the Col1a1 promoter rescued the defects mineralization as well as the defects in the dentinal tubules and third molar development. In contrast, re-expression in mature odontoblasts, using the Dspp promoter, produced only a partial rescue of the mineralization defects. These data suggest that DMP1 is a key regulator of odontoblast differentiation, formation of the dentin tubular system and mineralization and its expression is required in both early and late odontoblasts for normal odontogenesis to proceed.

As the rapid development of mobile communication technology, smartphones have become indispensable elements in our daily life. Particularly, the increasingly rich smartphone applications (apps) bring great convenience to people while the defects generated in app designing and coding may pose unexpected threats to users. In this paper, we focus on the issue of data leakage between the app client and server. By analyzing the vulnerabilities of client-to-server communication and eavesdropping on the session data, we implement spoofing attack on a popular music app client. Two experiments are introduced in details: downloading songs freely by means of bypassing the payment mechanism and deceiving user into installing malware. In addition, the countermeasures are also provided.