Jixin Ding

Aberrant Expression of Functional BAFF-System Receptors by Malignant B-Cell Precursors Impacts Leukemia Cell Survival

Despite exhibiting oncogenic events, patients leukemia cells are responsive and dependent on signals from their malignant bone marrow (BM) microenvironment, which modulate their survival, cell cycle progression, trafficking and resistance to chemotherapy. Identification of the signaling pathways mediating this leukemia/microenvironment interplay is critical for the development of novel molecular targeted therapies. We observed that primary leukemia B-cell precursors aberrantly express receptors of the BAFF-system, BAFF-R, BCMA, and TACI. These receptors are functional as their ligation triggers activation of NF-κB, MAPK/JNK, and Akt signaling. Leukemia cells express surface BAFF and APRIL ligands, and soluble BAFF is significantly higher in leukemia patients in comparison to age-matched controls. Interestingly, leukemia cells also express surface APRIL, which seems to be encoded by APRIL-δ, a novel isoform that lacks the furin convertase domain. Importantly, we observed BM microenvironmental cells express the ligands BAFF and APRIL, including surface and secreted BAFF by BM endothelial cells. Functional studies showed that signals through BAFF-system receptors impact the survival and basal proliferation of leukemia B-cell precursors, and support the involvement of both homotypic and heterotypic mechanisms. This study shows an unforeseen role for the BAFF-system in the biology of precursor B-cell leukemia, and suggests that the target disruption of BAFF signals may constitute a valid strategy for the treatment of this cancer.

The Role of MDM2 in Promoting Genome Stability versus Instability

In cancer, the mouse double minute 2 (MDM2) is an oncoprotein that contributes to the promotion of cell growth, survival, invasion, and therapeutic resistance. The impact of MDM2 on cell survival versus cell death is complex and dependent on levels of MDM2 isoforms, p53 status, and cellular context. Extensive investigations have demonstrated that MDM2 protein–protein interactions with p53 and other p53 family members (p63 and p73) block their ability to function as transcription factors that regulate cell growth and survival. Upon genotoxic insults, a dynamic and intricately regulated DNA damage response circuitry is activated leading to release of p53 from MDM2 and activation of cell cycle arrest. What ensues following DNA damage, depends on the extent of DNA damage and if the cell has sufficient DNA repair capacity. The well-known auto-regulatory loop between p53-MDM2 provides an additional layer of control as the cell either repairs DNA damage and survives (i.e., MDM2 re-engages with p53), or undergoes cell death (i.e., MDM2 does not re-engage p53). Furthermore, the decision to live or die is also influenced by chromatin-localized MDM2 which directly interacts with the Mre11-Rad50-Nbs1 complex and inhibits DNA damage-sensing giving rise to the potential for increased genome instability and cellular transformation.

Abstract 3180: Preclinical validation of EZH2 as a therapeutic target in pediatric Ewing's sarcoma

Disease-free survival in relapsed Ewing9s Sarcoma Family of Tumors (ESFT) has not improved over the past 25 years. Current standard-of-care (SOC) agents result in 70% survival in patients with localized ESFT; however, for relapsed patients the survival rates remain between 15-20%. Approximately 85% of ESFTs have the chromosomal translocation t(11;22)(q24;q12) which encodes for the oncogenic EWS/FL1 fusion protein. The EWS/FL1 functions as a potent transcription factor leading to the dysregulated expression of genes that promote and maintain tumorigenesis. A major epigenetic regulator that is a downstream target of EWS/FL1 is the enhancer of Zeste Homolog 2 (EZH2). EZH2 is the catalytic component of the polycomb repressor complex 2 (PRC2). Notably, it is overexpressed in ESFT and maintains tumor oncogenicity by tri-methylating histone 3 lysine 27 (H3K27me3) to modulate gene expression. Genome and transcriptome data obtained by the Pediatric Precision Genomics Program at Riley Hospital for Children at Indiana University Health (IUH) indicate that EZH2 is expressed at high levels in ESFT biopsies. Additionally, other groups have reported that high levels of EZH2 protein in ESFT and other cancers correlate with increased chemoresistance to SOC therapy. We are testing tested the hypothesis that EZH2 contributes to chemoresistance in ESFT by regulating critical growth and survival genes. In addition, we are investigating if pharmacological inhibition of EZH2 will enhance sensitivity to the cytotoxic effects of SOC agents. Pediatric primary and relapsed ESFT cell lines and ESFT xenografts were validated for the EWS/FLI, EZH2, and H3K27me3 signatures. In vitro- and in vivo-pharmacodynamic studies of EZH2 inhibition via tazemetostat were conducted to optimize dosing effect. In ESFT cell lines (TC71, A673, CHLA-9, and CHLA-10), tazemetostat dose-response experiments indicated a significant reduction of H3K27me3 by one day post-treatment which was either sustained or completely blocked by 7-days post-treatment compared to vehicle treated (p Citation Format: Pankita H. Pandya, Barbara Bailey, Adily E. Elmi, Heather R. Bates, Courtney N. Hemenway, Anthony L. Sinn, Khadijeh Bijangi-Vishehsaraei, M. Reza Saadatzadeh, Harlan E. Shannon, Jixin Ding, Mark S. Marshall, Michael J. Ferguson, Lijun Cheng, Lang Li, Mary E. Murray, Jamie L. Renbarger, Karen E. Pollok. Preclinical validation of EZH2 as a therapeutic target in pediatric Ewing9s sarcoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3180.

Abstract A49: Differential expression of the complement regulatory protein CD55 in wildtype and mutant p53 Glioblastoma and Ewing's Sarcoma

Background/Objectives: Solid tumors such as Glioblastoma (GBM) and Ewing9s Sarcoma result in relapse or refractory disease due the tumor9s ability to develop resistance to anti-cancer therapies. One mechanism by which solid tumors such as GBM and Ewing9s Sarcoma, circumvent the immuno-editing process and result in resistance to treatments is by the up-regulation of membrane-bound complement regulatory proteins (mCRPs) CD46, CD55, and CD59. While it has been reported that mCRPs are up-regulated in pediatric liquid tumors and influence the efficacy of monoclonal antibody treatments, regulation of mCRP expression in solid tumors has not been explored in detail. To delineate potential mechanisms regulating expression of mCRPs, we first screened wildtype (wt-p53) or mutant p53 solid tumor cell lines for mCRP expression. Our preliminary data suggest that p53 status correlates with CD55 but not CD46 or CD59 expression. These studies may serve to be the foundation for potentially recognizing the mCRPs as immune biomarkers in pediatric and adult solid tumors, ultimately, resulting in the development of novel immunotherapies for improved clinical outcomes and patient care. Design/Methods: Adult GBM and pediatric Ewing9s Sarcoma cell lines that are wildtype p53 (GBM10 and CHLA-9) or mutant p53 (GBM-43 and CHLA-10) were exploited for in vitro studies. The GBM-43 was generated from a primary GBM while the GBM-10 from a patient with recurrent GBM. The paired Ewing9s Sarcoma cell lines, CHLA-9 and CHLA-10, were generated from the same patient at primary diagnosis and at relapse respectively. Western blot and qPCR were used to confirm expression and mutational status of p53. mCRP expression was evaluated using RT-PCR and flow cytometry. Results: All cancer cell lines expressed p53 to varying degrees. No significant difference was observed in the expression of CD46 and CD59 among the wt-p53 and mutant p53 glioblastoma cell lines. Mutant p53 cell lines from glioblastoma and Ewing9s sarcoma had increased CD55 transcript levels compared to their wildtype counterpart cell lines where the transcripts were undetectable. In addition, flow cytometry data revealed increased expression of the mCRP, CD55, in mutant p53 glioblastoma (GBM-43) versus wt-p53 (GBM-10) cells (p Conclusion: These preliminary findings highlight the importance of further investigating the role of mCRPs on wt-p53 and mutant p53 cell lines. Studies are in progress to determine if expression of CD55 is regulated by p53 status. Understanding how this critical mCRP is regulated in solid tumors will be important for immune biomarker development as well as useful in guiding the use of antibody-based therapeutic approaches in solid tumors. Citation Format: Pankita H. Pandya, Reza Saadatzadeh, Jixin Ding, Barbara Bailey, Sydney Ross, Mary E. Murray, Jamie L. Renbarger, Karen E. Pollok. Differential expression of the complement regulatory protein CD55 in wildtype and mutant p53 Glioblastoma and Ewing9s Sarcoma. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2016 Oct 20-23; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2017;5(3 Suppl):Abstract nr A49.

Abstract 4592: Complement regulatory protein expression in solid tumors: implications for resistance to antibody-mediated immunotherapy

Background: Resistance to anti-cancer therapies results in relapsed/refractory disease of Glioblastoma (GBM) and Ewing’s Sarcoma. Up-regulation of membrane-bound complement regulatory proteins (mCRPs) CD46, CD55, and CD59 can enable solid tumors to confer resistance to antibody-mediated immunotherapy by preventing complement and antibody-dependent cytotoxicity. mCRPs’ inhibitory role in monoclonal antibody treatments for liquid tumors have been reported, but their role and regulation in solid tumors has not been explored. In the context of refractory tumors, others have reported that vascular endothelial growth factor-A (VEGF-A) can induce mCRP expression in endothelial cells. Notably, p53 mutational status induces VEGF-A and its receptor (VEGFR2) in breast cancer cell lines. To investigate potential links among p53 status, VEGF-A, and mCRP, we screened wildtype (wt-p53) and mutant p53 solid tumor cell lines for mCRP expression and VEGF-A secretion. Our data suggest that p53 mutational status is associated with expression of CD55 and VEGF-A secretion. These studies provide foundation for potentially recognizing mCRPs as immune biomarkers in solid tumors, ultimately, resulting in development of novel immunotherapies for improved clinical outcomes. Methods: Pediatric Ewing’s sarcoma (CHLA9 and CHLA10) and adult GBM (GBM10 and GBM43) cell lines differing in p53 status were selected for in vitro studies. GBM43 originates from a primary GBM, while GBM10 is from a recurrent GBM patient. Ewing’s Sarcoma cell lines, CHLA9 and CHLA10, were generated from the same patient at primary diagnosis and at relapse respectively. Western blot, and sequencing confirmed the expression and p53 mutational status. mCRP expression was evaluated using RT-PCR and flow cytometry. Milliplex platform assessed VEGF-A expression in cell supernatants. Results: Whole genome sequencing data confirmed p53 mutations in all cell lines. CHLA9 and GBM10 harbor wt-p53. CHLA10 cells have p53 deletion and GBM43 cells have a F270C p53 mutation in both alleles CD55 transcripts were undetected in wt-p53 lines (CHLA9 and GBM10), but CD55 transcripts were increased in mutant/deleted p53 lines (CHLA10 and GBM43). Flow cytometry data show increased CD55 expression in mutant p53 glioblastoma (GBM43) versus wt-p53 (GBM10) cells (p Conclusion: These findings highlight the importance of further investigating role of VEGF-A in regulating mCRPs in wt-p53 versus mutant p53 solid tumor cell lines. Elucidating mechanisms for mCRP regulation is critical for immune biomarker development and in facilitating the use of antibody-based therapeutic approaches for solid tumors. Citation Format: Pankita Hemant Pandya, M. R. Saadatzadeh, Jixin Ding, Barbara Bailey, Sydney Ross, Khadijeh Bijangi-Vishehsaraei, Mary E. Murray, Karen E. Pollok, Jamie L. Renbarger. Complement regulatory protein expression in solid tumors: implications for resistance to antibody-mediated immunotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4592. doi:10.1158/1538-7445.AM2017-4592

Abstract A26: Inhibition of MDM2 and AKT signaling networks synergize to activate Forkhead box O-class transcription factors and promote cell death in mutant p53 GBM cells

A multi-targeted approach will be necessary to eradicate glioblastoma multiforme (GBM) cells due to the immense genetic heterogeneity associated with GBM. Mouse double minute-2 (MDM2) regulates multiple signaling pathways and is a promising therapeutic target in GBM. In wild type (wt) p53 cells, MDM2 binds to wtp53, ubiquitinates it, and negatively regulates p53-mediated downstream events. In wtp53 and mutant (mt) p53 cells, MDM2 binds to and sequesters p73α thereby blocking p73α-mediated signaling. Our objective in the present studies was to determine if the p73α-MDM2 axis could be exploited to increase death of mtp53 GBM cells. We utilized MDM2 antagonists nutlin3a or RG7112 to block protein-protein interactions between MDM2-p53 and MDM2-p73α. In a panel of GBM cell lines, TMZ resistance was reduced in both wt53 and mt53 cells in the presence of MDM2 antagonists. In mtp53 cells, siRNA knockdown of p73α indicated that sensitivity to treatment was dependent on p73α levels. Isobologram analysis indicated that while dose-ratios of TMZ to MDM2 antagonists were additive to synergistic in inhibiting growth of wtp53 GBM cells, this was not the case in mtp53 GBM cells (SF118, GBM43, gain-of-function-mtp53 R273H U373 and MHBT32). Analysis of intracellular targets in mtp53 GBM cells exposed to TMZ and MDM2 antagonists indicated that p73α and MDM2 expression increased by 24 hours post-treatment. In addition, AKT activity was increased or sustained in mtp53 GBM cells following treatment with TMZ in the absence or presence of MDM2 antagonists. Since increased AKT activity may render cells resistant to therapy, the AKT inhibitor GDC0068 was evaluated in combination with TMZ and RG7112. As a measure of AKT-downstream target modulation, phosphorylation status of the Forkhead box O-class (FoxO) transcription factors (TFs) was determined. In the non-phosphorylated state, FoxO TFs upregulate expression of proteins involved in cell-death pathways. While phospho-FoxO1/FoxO3a TFs were increased in TMZ/RG7112-treated mtp53 GBM cells compared to controls, it was decreased in GDC0068-, TMZ/GDC0068- and TMZ/RG7112/GDC0068-treated mtp53 GBM cells which is consistent with inactivation of AKT and activation of FoxO TFs. Isobologram analysis of mtp53 GBM cell growth indicated that combination RG7112 and GDC0068 inhibited growth in a synergistic manner even in the absence of TMZ. For in vivo studies, an intermittent dosing regimen of TMZ/RG7112/GDC0068 was developed to avoid normal tissue toxicity. GBM43 flank tumor growth was significantly inhibited in mice with tumors treated with RG7112/GDC0068 and inhibited to a larger extent by the triple combination TMZ/RG7112/GDC0068 compared to vehicle and single-agent exposure (n=9-10 mice per group; single agent vs GDC0068/RG7112 or TMZ/RG7112/GDC0068, p in vivo with an acceptable toxicity profile. Citation Format: Mohammad Reza Saadatzadeh, Haiyan Wang, Jixin Ding, Barbara J. Bailey, Eva Tonsing-Carter, Shanbao Cai, Nimita Dave, Harlan E. Shannon, Aaron Cohen- Gadol, Karen E. Pollok. Inhibition of MDM2 and AKT signaling networks synergize to activate Forkhead box O-class transcription factors and promote cell death in mutant p53 GBM cells. [abstract]. In: Proceedings of the AACR Special Conference: Advances in Brain Cancer Research; May 27-30, 2015; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2015;75(23 Suppl):Abstract nr A26.

Abstract B32: First-in-class Ref-1 redox inhibitors for the multipathway targeting of survival signals for relapsed childhood acute lymphoblastic leukemia

Acute lymphoblastic leukemia (ALL) is the most frequent pediatric cancer and, despite significant treatment advances, relapsed ALL remains the second leading cause of childhood death. In T-cell ALL, more than 10% of the patients show poor response to frontline therapy and about a third of the patients in remission develop recurrence of their leukemia. Importantly, effective, curative therapies are lacking for children with relapse or refractory disease. Our strategy for T-ALL was to develop agents that target master molecular regulators controlling the activity of multiple complementary, non-recurrent signaling pathways, integrating oncogenic signals and microenvironment cues. Ref-1/APE1 (Ref-1) is a multi-function protein that exerts redox control of multiple transcription factors (TFs), regulating their DNA binding and downstream transcriptional programs. These include TFs playing important roles in T-cell ALL, namely NF-κB, AP-1 and STAT3, the latter of which we have shown recently to be required for T-cell leukemogenesis. We showed that Ref-1 is expressed by leukemia T-cells in the malignant bone marrow (BM), and that its expression is increased in drug-resistant T-ALL cells. Analyses of multiple leukemia transcriptome databases showed significant increased expression of Ref-1 in T-cell ALL specimens, as well as of other genes in the Ref-1/SET molecular axis. Molecular and functional studies showed that disruption of Ref-1 redox function markedly inhibits leukemia T-cell survival and proliferation, triggering molecular changes promoting cell apoptosis. We identified three new small molecule chemical entities - APX2007, APX2009 and APX2032, that significantly inhibit the reduction of Ref-1 and the DNA binding of Ref-1-regulated TFs as assessed by EMSA. These compounds (cpds) are significantly more potent than a previously identified Ref-1 redox antagonist, E3330. Functional studies showed that all three APX cpds markedly inhibit leukemia cell survival. Potent inhibition of tumor cell viability was seen in primary cells from ALL patients, relapsed T-ALL, and cells from a murine model of Notch-induced leukemia. Blockade of Ref-1 redox triggers significant leukemia cell apoptosis, and correlates with down-regulation of survival genes regulated by the Ref-1 ‘targets’ STAT3 and NF-kB. Blockade of Ref-1 redox by the APX cpds markedly inhibits the viability of drug- resistant T-ALL cells, with an antitumor efficacy comparable to chemotherapy-sensitive leukemia cells. This is significant since glucocorticoid-resistance is predictive of ALL relapse, and glucocorticoid-resistant leukemia T-cells show reduced sensitivity to inhibitors of other leukemia-associated signaling pathways (as PI3K/Akt, mTOR). Preliminary safety studies in mice using a clinical formulation demonstrated the systemic administration of the APX cpds do not result in acute adverse reactions or significant hematological toxicities. Importantly, studies in a xenograft model of glucocorticoid-, doxorubicin-resistant human T-ALL showed that treatment with the APX cpds result in significant decrease in leukemia blasts in the peripheral blood and in the BM. In summary, we developed novel, first-in-class inhibitors of Ref-1, which show acceptable in vivo PK and toxicity, and that potently inhibit T-ALL, including patient9s specimens and drug-resistant leukemia T-cells. These new chemical entities target a unique molecular regulator, as Ref-1 redox function controls multiple TFs involved in leukemogenesis and disease progression. Studies are underway to further define the therapeutic efficacy and long-term remission potential of ApeX compounds in animal models of T-ALL, including a model of leukemia recurrence post-frontline chemotherapy. These studies should support the selection of a candidate for development and progression to clinical trials in pediatric patients with refractory, relapsed ALL. Citation Format: Angelo A. Cardoso, James H. Wikel, Jixin Ding, April M. Reed, Meihua Luo, Mark R. Kelley. First-in-class Ref-1 redox inhibitors for the multipathway targeting of survival signals for relapsed childhood acute lymphoblastic leukemia. [abstract]. In: Proceedings of the AACR Special Conference on Pediatric Cancer at the Crossroads: Translating Discovery into Improved Outcomes; Nov 3-6, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;74(20 Suppl):Abstract nr B32.