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Neuroblastoma (NB) is the most common extracranial solid malignancy in children. As leading regulators of inflammation and tumor progression, tumor associated macrophages (TAMs) have gained major interest as immunotherapeutic targets in NB. Hence targeting or “re-educating” tumorigenic macrophages towards an immunostimulatory phenotype might improve responses to immunotherapy in this childhood cancer. Spleen tyrosine kinase (Syk) has recently been identified by our group as a novel immune-oncology target. We found that Syk is abundantly present in TAMs infiltrated in MYCN and non-MYCN amplified neuroblastoma tumors. Furthermore, genetic deletion or pharmacological inhibition of Syk markedly impaired neuroblastoma tumor growth. This effect was facilitated by macrophages that became immunogenic in the absence of Syk, skewing the suppressive tumor microenvironment towards immunostimulation and activating antitumor immune responses. Moreover, combining Syk inhibitor, R788 along with anti-PDL1 mAb and radiation provided a synergistic effect leading to complete tumor regression and durable anti-tumor immunity in aggressive MYCN-and non-MYCN-driven murine neuroblastoma tumor models. Collectively, our findings demonstrate the importance of Syk inhibitor, R788, in enhancing the anti-tumor immune responses in NB and support the clinical evaluation of R788 either alone or together with radiation and immune check point inhibitors as a potential treatment strategy for NB. Citation Format: Deepak Rohila, In Hwan Park, Timothy Pham, Riley Jones, Pablo Tamayo, Alice Yu, Andrew Sharabi, Shweta Joshi. Targeting Syk reprograms tumor-associated macrophages and enhances responses to immune checkpoint blockade and radiation therapy in high-risk neuroblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3246.

Background: Arginine (Arg) deprivation is a promising therapeutic approach for tumors with low argininosuccinate synthetase 1 (ASS1) expression. However, its efficacy as a single agent therapy needs to be improved as resistance is frequently observed. Methods: A tissue microarray was performed to assess ASS1 expression in surgical specimens of pancreatic ductal adenocarcinoma (PDAC) and its correlation with disease prognosis. An RNA-Seq analysis examined the role of ASS1 in regulating global gene transcriptome. A high throughput screen of FDA-approved oncology drugs identified synthetic lethality between histone deacetylase (HDAC) inhibitors and Arg deprivation in PDAC cells with low ASS1 expression. We examined HDAC inhibitor panobinostat (PAN) and Arg deprivation in a panel of human PDAC cell lines, in ASS1-high and -knockdown/knockout isogenic models, in both anchorage-dependent and -independent cultures, and in multicellular complex cultures that model the PDAC tumor microenvironment. We examined the effects of combined Arg deprivation and PAN on DNA damage and the protein levels of key DNA repair enzymes. We also evaluate the efficacy of PAN and ADI-PEG20 (an Arg-degrading agent currently in Phase II clinical trials) in xenograft models with ASS1-low and -high PDAC tumors. Results: Low ASS1 protein level is a negative prognostic indicator in PDAC. Arg deprivation in ASS1-deficient PDAC cells upregulated asparagine synthetase (ASNS) which redirected aspartate (Asp) from being used for de novo nucleotide biosynthesis, thus causing nucleotide insufficiency and impairing cell cycle S-phase progression. Comprehensively validated, HDAC inhibitors and Arg deprivation showed synthetic lethality in ASS1-low PDAC cells. Mechanistically, combined Arg deprivation and HDAC inhibition triggered degradation of a key DNA repair enzyme C-terminal-binding protein interacting protein (CtIP), resulting in DNA damage and apoptosis. In addition, S-phase-retained ASS1-low PDAC cells (due to Arg deprivation) were also sensitized to DNA damage, thus yielding effective cell death. Compared to single agents, the combination of PAN and ADI-PEG20 showed better efficacy in suppressing ASS1-low PDAC tumor growth in mouse xenograft models. Conclusion: The combination of PAN and ADI-PEG20 is a rational translational therapeutic strategy for treating ASS1-low PDAC tumors through synergistic induction of DNA damage. Citation Format: Stephanie S Kim, JING CUI, Shili Xu, Soumya Poddar, Thuc M. Le, Luyi Li, Nanping Wu, Alexandra Moore, Lei Zhou, Alice Yu, Amanda M. Dann, Irmina A. Elliott, Evan R. Abt, Woosuk Kim, David W. Dawson, Caius G. Radu, Timothy R. Donahue. Histone deacetylase inhibition is synthetically lethal with arginine deprivation in pancreatic cancers with low argininosuccinate synthetase 1 expression [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 554.

Purpose: Prognosis after radical cystectomy for bladder cancer can be unpredictable. There is increasing evidence that the presence of cytotoxic T lymphocytes correlates with survival. The Immunoscore, a new approach to classification of cancer using the number, type and distribution of immune cells, is an independent predictor for survival in colon cancer. Our objective was to evaluate the prognostic impact of lymphocyte distribution and the Immnoscore in bladder cancer. Methods: Cystectomy sections with core tumor (CT) involvement and identifiable invasive margin (IM) were selected and stained for CD3+ and CD8+ lymphocytes. Three non-contiguous areas of highest lymphocyte density were selected from both CT and IM. The number of lymphocytes in each area was used to calculate the Immunoscore based on previously defined criteria. Kaplan-Meier curves were used to determine differences between Disease-free Survival (DFS) and Overall Survival (OS). Multivariate Cox proportional hazards model was used to determine hazard ratios. Results: Total of 67 patients who had cystectomy for invasive bladder cancer were included in the study. High concentration of CD8+ lymphocytes in the tumour margin was associated with better DFS (P=0.005) and OS (P=0.03). Similar results were found for CD3+ lymphocytes with regards to DFS (P=0.05). A higher Immunoscore was also associated with better DFS (P=0.04). On multivariate analysis, CD8+ in the invasive margin was independently associated with better prognosis. Conclusions: The host’s own immune system plays a valuable role in clinical outcome. Our data suggests that a strong immune response in the tumor margin is independently associated with better prognosis. Citation Format: Jose J. Mansure, Alice Yu, Shraddha Solanki, Ana B. Dias, Miguel M. Burnier, Fadi Brimo, Wassim Kassouf. Cytotoxic T lymphocyte CD3+, CD8+, and immunoscore as prognostic marker in patients after radical cystectomy [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 1643. doi:10.1158/1538-7445.AM2017-1643

Beckwith-Wiedemann Syndrome (BWS) is a cancer predisposition syndrome that affects at least 1 in 10,500 children. Up to 25% of children with BWS develop tumors, primarily Wilms tumor and hepatoblastoma. BWS is due to genetic or epigenetic changes that affect imprinted loci on chromosome 11 and these same changes are also found in other types of cancer. There are no cell-based models of BWS and most mouse models do not recapitulate the tumor phenotype. To understand more about the mechanisms leading to tumor formation in BWS, we developed the first human cell-based model of BWS. Human induced pluripotent stem cell (iPSC) models are commonly used to study disease mechanisms in tissue types that are not normally accessible for study. In the case of BWS, we plan to use such models to study how the genetic and epigenetic changes in BWS lead to tumor formation in hepatic and renal cells. Using skin fibroblasts from four BWS patients, we derived the first iPSC models of BWS. Prior to iPSC derivation, we characterized different tissues available from these four patients, and demonstrated genetic mosaicism in different tissue types, including blood, skin, and pancreas. During the derivation process, we demonstrated that both normal and BWS iPSC lines could be derived from the same patient fibroblast sample and the number of clones of each type from each sample approximated the initial level of mosaicism in the original sample. For each patient, these lines are isogenic except for the BWS critical region. The BWS and isogenic normal iPSCs were characterized for pluripotency markers and demonstrated to have normal karyotypes. Following this analysis, BWS and isogenic normal lines were characterized extensively for DNA methylation at specific imprinted loci in both early passage and extended culture. Methylation analysis was performed by both pyrosequencing and COBRA assays. Methylation was maintained at some imprinted loci but not at others in extended culture. Importantly, relatively stable methylation levels were observed at the BWS critical imprinted regions (H19/IGF2 and KvDMR), regardless of methods of reprogramming, indicating a relatively stable state of DNA methylation in this region. Additionally, normal methylation was seen at the SNRPN locus. In contrast, another imprinted locus, IG/MEG3, displayed abnormal hypermethylation in iPSCs. These data indicate that reprogramming and extended culture of iPSCs can affect stability of DNA methylation at certain imprinted loci. Therefore caution should be used in interpreting studies using iPSCs as these aberrant methylation states at imprinted loci can affect the downstream functionality of iPSC models. BWS iPSCs and isogenic controls with normal methylation will be used for further study of the mechanism of tumor formation in BWS. Note: This abstract was not presented at the meeting. Citation Format: Stella K. Hur, Joanne L. Thorvaldsen, Suhee Chang, Carolyn Lye, Alice Yu, Monserrat C. Anguera, Marisa S. Bartolomei, Jennifer M. Kalish. Epigenetic challenges in derivation of the first cell-based model of Beckwith-Wiedemann Syndrome [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 1936. doi:10.1158/1538-7445.AM2017-1936

Introduction Metabolic reprogramming is a cancer hallmark that is not fully understood. Moreover, increasing evidence suggests that metabolic dysregulation is histology-specific, particularly in the case of non-small cell lung cancer (NSCLC). In this study, we investigated the differences in glucose metabolism between the two major histology subtypes of NSCLC, namely adenocarcinoma (AD) and squamous cell carcinoma (SQ). We explored transcriptomics in association with FDG-PET SUVmax (maximum standardized uptake value), a common clinical marker of glucose uptake in cancer. Methods We identified metabolically-associated differentially expressed genes (DEGs) by histology subtypes using RNAseq data from resected tumors in our study cohort of 127 NSCLC patients (96 AD and 31 SQ) and validated the DEGs on The Cancer Genome Atlas. We also identified gene-enriched cellular processes correlated with SUVmax separately for AD and SQ. We validated our findings in public domain cell line microarray data. In a separate cohort of 40 NSCLC patients (20 patients overlap with study cohort) whose tumors were sorted by cell type (cancer, endothelial, fibroblast and immune cells), we identified specific cell types where the genes of interest were expressed. Results Metabolically-associated DEGs in AD were enriched in the hexosamine biosynthesis pathway (HBP), which is involved in glycosylating proteins related to invasion. SUVmax correlated genes in AD were enriched for epithelial-mesenchymal transition (EMT) and extracellular matrix remodeling. In particular, GFPT2 (the rate-limiting gene in HBP) and 4 EMT-related glycoprotein genes were highly correlated with SUVmax, suggesting potency of HBP for EMT related glucose metabolic reprogramming. We validated the findings on cell line microarray data (GSE49644) before and after TGF-β induced EMT, showing that many genes in HBP increased after induced EMT, while genes in the glycolysis pathway and the pentose phosphate pathway (PPP) were unchanged or reduced. HBP genes and many SUVmax correlated EMT genes were most highly expressed in tumor-associated fibroblasts compared to other cells in the tumor microenvironment. In SQ, metabolically-associated DEGs were enriched in the glycolysis pathway and PPP. SUVmax correlated genes were enriched for cell development. Conclusion An integrative analysis of PET-RNAseq showed that in AD glucose uptake was related to invasion through metabolic reprogramming of HBP. In SQ, glucose uptake was associated with glycolysis and higher proliferation potential. Our work confirms that metabolic dysregulation in NSCLC is histology-specific and extends the finding to identify cellular processes associated with FDG uptake that differ by histology as well. Understanding histology-specific differences of tumor metabolism provides new insights into tumor behavior that can have implications for anti-cancer therapy. Citation Format: Weiruo Zhang, Gina Bouchard, Alice Yu, Majid Shafiq, Mehran Jamali, Joseph Shrager, Kelsey Ayers, Viswam S. Nair, Andrew Gentles, Maximilian Diehn, Andrew Quon, Sandy Napel, Sylvia Plevritis. FDG uptake in human lung adenocarcinoma associated with invasion through the hexosamine biosynthesis pathway [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 4403. doi:10.1158/1538-7445.AM2017-4403

Telomere maintenance and telomerase reactivation are near universal features of human hepatocellular carcinoma (HCC), yet the shorter telomeres and highly abnormal cytogenetic profiles of HCC suggest that telomere erosion and dysfunction may be operative during the formative stages of tumorigenesis. Previous studies have established that the cancer-enhancing or suppressing impact of telomere dysfunction is highly dependent on several parameters including cell type, tumor stage, and p53 status. Here, to understand better the pathogenetic role of telomere dysfunction in the initiation and progression in human HCC, we have used three mechanistically distinct liver cancer-prone model systems (urokinase plasminogen activator transgenic mice, carbon tetrachloride exposure, and diethylnistrosamine treatment) in the context of successive generations of telomerase-deficient mice null for the telomerase RNA component, mTERC. Across all of the HCC model systems, telomere dysfunction suppressed both the incidence and growth of HCC lesions, a trend that mirrored the level of intratumoral proliferative arrest and apoptosis. On the histological level, telomere dysfunction was associated with a significant increase in the number of early stage neoplastic lesions and a reciprocal decline in the occurrence of high-grade malignancies. These genetic data in the mouse indicate that telomere dysfunction exerts an opposing role in the initiation versus progression of HCC and provide a framework for understanding the intimate link among chronic liver disease, chromosomal instability, and increased HCC in humans.

Increased expression of intracellular thioredoxin has been implicated in the inhibition of apoptosis and in a decrease in the sensitivity of the malignancies to drug-induced apoptosis. In the present studies, we analyzed expression of thioredoxin in samples from 28 children with T-cell acute lymphoblastic leukemia and analyzed their sensitivity toward inhibition of thioredoxin expression. Thioredoxin was expressed in variable amounts. Higher expression was associated with higher WBC counts. Exogenously added thioredoxin stimulated proliferation of clonogenic cells among the T-cell acute lymphoblastic leukemia samples expressing relatively lower levels of intracellular thioredoxin, whereas there was no effect on the clonogenic cells expressing high levels of thioredoxin. In addition, there was differential sensitivity of the leukemia clonogenic cells toward 1-methylpropyl 2-imidazolyl disulfide, an inhibitor of thioredoxin expression, as compared with normal hematopoietic progenitors. This suggests the possibility of using this approach for treatment. Because overexpression of thioredoxin is associated with resistance to many anticancer drugs, the inhibition of thioredoxin expression may overcome this drug resistance and probably sensitize leukemia cells to other chemotherapeutic agents.

Exposure to ultraviolet (UV) light is the major etiologic factor leading to the development of cutaneous squamous and basal cell carcinoma and is also a risk factor for melanomas. UV elicits inflammation, epidermal hyperplasia and changes in the expression of numerous genes associated with proliferation and differentiation. Curcumin has been shown to exert an anti-inflammatory activity, interfere with multiple cell signaling pathways, and possess potential preventive effect against various cancers. The purpose of this study was to determine whether curcumin can protect UV-induced skin damage in hairless SKH-1 mice. Oral or topical application of curcumin prior to, or immediately after, UV irradiation resulted in a very strong protective effect against UV-induced thymine dimer expression in epidermis, accounting for 69-74% inhibition (p < 0.00). Curcumin treatment also resulted in upregulation of p53 and p21/Cip1 protein levels by 33-41% (p < 0.005) and 46-50% (p < 0.005), respectively. Oral or topical application of curcumin prior to, or immediately after UV irradiation strongly decreased the expression of proliferative cell nuclear antigen by 47- 56% (p < 0.005). In addition, curcumin treatment significantly suppressed UV-induced apoptosis as measured by TUNEL assays (p < 0.005). Furthermore, curcumin treatment also significantly inhibited NF-κB, cyclooxygenase-2 (COX-2), prostaglandin E2 (PGE2), and nitric oxide (NO) levels. Our results suggest that curcumin possesses strong protective effect against UV-caused damage in epidermis. Taken together, these results provide a focus for the rational development of curcumin as a novel chemopreventive agent against UV-induced human skin cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 503.

We have devised methods facilitating the establishment of continuous cultures of T-cell blasts from patients with acute lymphoblastic leukemia of T-cell type at diagnosis. The cultured cells closely resemble those of the patients at the time of diagnosis with respect to surface markers, karyotype, and T-cell receptor gene rearrangements. Cultured T-cell acute lymphoblastic leukemia (diagnosis) cells (a) are lymphocytes with a convoluted nucleus; (b) have doubling times of 24-48 h; (c) are dependent for growth on interleukin 2; (d) are reverse transcriptase negative; (e) do not form colonies in methyl cellulose; and (f) are clonal with respect to T-cell receptor beta chain rearrangements. Three T-cell acute lymphoblastic leukemia cultures had a normal diploid karyotype, and one had a 6q- deletion which was also present at the time of diagnosis.

The in vitro effects of deoxyadenosine and an adenosine deaminase inhibitor, deoxycoformycin, on the synthesis of DNA and the metabolism of purines were investigated in human leukemic T-cells. In the presence of 10 microM deoxycoformycin, the synthesis of DNA was completely inhibited by concentrations of deoxyadenosine of 10 microM or greater. In contrast, the synthesis of DNA in normal bone marrow cells was not inhibited in the presence of up to 20 microM deoxycoformycin and up to 10 microM deoxyadenosine. Following incubation of leukemia T-cells with deoxycoformycin and deoxyadenosine, there was a significant rise in the concentration of deoxyadenosine 5'-triphosphate which was accompanied by reductions in the concentrations of adenosine 5'-triphosphate and guanosine 5'-triphosphate, as revealed by high-pressure liquid chromatographic analysis. The effects of deoxycoformycin on T-cell leukemia were examined in vivo. A patient with acute T-cell leukemia in the terminal stage received five daily injections of 250 micrograms of deoxycoformycin per kg. Among the noted changes, most prominent was the drop in the leukocyte count. Initially, the cell count rose from 7,200 cells/microliters on Day 1 to 120,000 cells/microliters on Day 3. On Day 5, the cell count began to decline and reached a nadir of 600 cells/microliter on Day 10. The leukocyte count remained below 1,000 cells/microliter through Day 12. The reduction in cell count was preceded by a decline in the incorporation of [3H]thymidine in the cells, which dropped to negligible amount by Day 7. The other prominent change was a decrease in adenosine deaminase activity in both red cells and leukemic cells. Adenosine deaminase activity of red cells dropped to 5% on Day 4, and that of leukemic cells dropped to 59% on Day 5. In addition, there were considerable alterations in the concentrations of purine metabolites which were characterized by a progressive reduction in the concentrations of total purine metabolites, especially adenosine 5'-triphosphate, and a transient rise in the concentrations of deoxyadenosine 5'-triphosphate, adenosine 5'-monophosphate, and adenosine 5-diphosphate. These findings suggest that treatment with deoxycoformycin may be of therapeutic value for T-cell leukemia. It may provide opportunities for studying the purine metabolism in T-leukemic cells which could lead to better approaches to treatment.