Search

Supplementary Figure 5. Assessment of citric acid cycle activity by protein expression analysis of PDHE1a.

Supplementary Table 1 - 7. Supplementary Table 1: Clinical and histopathological data associated with all spinal ependymomas analyzed in this study, along with molecular subgroup assignment; Supplementary Table 2: Table of all antibodies used in this study, their commercial sources, and dilutions used; Supplementary Table 3: Table of differentially expressed genes between spinal grade II and myxopapillary ependymoma; Supplementary Table 4: Summary of focal amplifications detected in spinal ependymoma by GISTIC2; Supplementary Table 5: Summary of focal deletions detected in spinal ependymoma by GISTIC2; Supplementary Table 6: List of significant pathways enriched by Gene Set Enrichment Analysis in spinal grade II ependymoma; Supplementary Table 7: List of significant pathways enriched by Gene Set Enrichment Analysis in spinal myxopapillary ependymoma

Supplementary Figure 1. Unsupervised consensus hierarchical clustering and non-negative matrix factorization of 35 spinal ependymoma gene expression signatures from a rank 2 to 4 classification.

Supplementary Figure 2. Summary of focal copy number alterations detected by GISTIC2 in spinal Grade II and Myxopapillary Ependymoma.

Supplementary Figure 3. Analysis of HIF-1a expression in Spinal Grade II versus Myxopapillary Ependymoma in additional to single sample GSEA analysis of HIF1a transcriptional targets.

Supplementary Figure 4. Metabolic protein expression analysis supporting Figure 4 along with lactate measurements in normal spinal tissue, Grade II spinal ependymoma, and myxopapillary ependymoma.

Purpose: Myxopapillary ependymoma (MPE) is a distinct histologic variant of ependymoma arising commonly in the spinal cord. Despite an overall favorable prognosis, distant metastases, subarachnoid dissemination, and late recurrences have been reported. Currently, the only effective treatment for MPE is gross-total resection. We characterized the genomic and transcriptional landscape of spinal ependymomas in an effort to delineate the genetic basis of this disease and identify new leads for therapy.Experimental Design: Gene expression profiling was performed on 35 spinal ependymomas, and copy number profiling was done on an overlapping cohort of 46 spinal ependymomas. Functional validation experiments were performed on tumor lysates consisting of assays measuring pyruvate kinase M activity (PKM), hexokinase activity (HK), and lactate production.Results: At a gene expression level, we demonstrate that spinal grade II and MPE are molecularly and biologically distinct. These are supported by specific copy number alterations occurring in each histologic variant. Pathway analysis revealed that MPE are characterized by increased cellular metabolism, associated with upregulation of HIF1α. These findings were validated by Western blot analysis demonstrating increased protein expression of HIF1α, HK2, PDK1, and phosphorylation of PDHE1A. Functional assays were performed on MPE lysates, which demonstrated decreased PKM activity, increased HK activity, and elevated lactate production.Conclusions: Our findings suggest that MPE may be driven by a Warburg metabolic phenotype. The key enzymes promoting the Warburg phenotype: HK2, PKM2, and PDK are targetable by small-molecule inhibitors/activators, and should be considered for evaluation in future clinical trials for MPE. Clin Cancer Res; 21(16); 3750–8. ©2015 AACR.

Supplementary Figure 6. A schematic of the glycolytic pathway in myxopapillary ependymoma illustrating deregulation of proteins and metabolites and possible targets for therapeutic intervention.

Supplementary Figure and Table Legends. Legends describing in detail the data shown in supplementary figures and tables pertaining to this manuscript.

KM curve of 100 cell injection, quantization of Ki67 and Casp3 staining, and self-renewal of TICs.

PDF file - 65K, Supplementary Figure 6. A schematic drawing of full length N1ICD in mouse, N1ICD allele analyzed in this study, and the ACTB-Notch1 allele previously published (Yang et al., 2004) and analyzed in Supplementary Figure 6.

Supplementary Figure Legends from Epigenetic States of Cells of Origin and Tumor Evolution Drive Tumor-Initiating Cell Phenotype and Tumor Heterogeneity

KM survival curve, quantitation of differentiated cells, and H&E of tumorsphere induced tumors.

IPA analysis.

PDF file - 133K, Supplementary Figure 3. Reduced p53 dose rescues precocious GFAP expression in the N1ICD;hGFAP-cre brains.

PDF file - 84K, Supplementary Figure 2. Reduced p53 dose rescues brain sizes in N1ICD;hGFAP-cre transgenic mice.

Genes that are differentially expressed among the subtypes.

FISH results from Ptch tumors that progressed to the GFI subtype during in vivo passaging.

While activation of the Notch pathway is observed in many human cancers, it is unknown whether elevated Notch1 expression is sufficient to initiate tumorigenesis in most tissues. To test the oncogenic potential of Notch1 in solid tumors, we expressed an activated form of Notch1 (N1ICD) in the developing mouse brain. N1ICD;hGFAP-cre mice were viable but developed severe ataxia and seizures, and died by weaning age. Analysis of transgenic embryo brains revealed that N1ICD expression induced p53-dependent apoptosis. When apoptosis was blocked by genetic deletion of p53, 30% to 40% of N1ICD;GFAP-cre;p53+/− and N1ICD;GFAP-cre;p53−/− mice developed spontaneous medulloblastomas. Interestingly, N1ICD-induced medulloblastomas most closely resembled the sonic hedgehog subgroup of human medulloblastoma at the molecular level. Surprisingly, N1ICD-induced tumors do not maintain high levels of the Notch pathway gene expression, except for Notch2, showing that initiating oncogenic events may not be decipherable by analyzing growing tumors in some cases. In summary, this study shows that Notch1 has an oncogenic potential in the brain when combined with other oncogenic hits, such as p53 loss, and provides a novel mouse model of medulloblastoma. Cancer Res; 73(17); 5381–90. ©2013 AACR.

PDF file - 79K

PDF file - 89K

PDF file - 158K, Supplementary Figure 1. Reduced p53 dose rescues apoptosis in N1ICD;hGFAP-cre brains.

PDF file - 199K, Supplementary Figure 5. Increased apoptosis and reduced proliferation of neural precursor cells in ACTB-N1ICD brains.

PDF file - 84K, Supplementary Figure 4. N1ICD induced medulloblastomas.

Supplementary Methods

H&E of tumor derived from injecting SMO;GFAP_cre tumor spheres.

A central confounding factor in the development of targeted therapies is tumor cell heterogeneity, particularly in tumor-initiating cells (TIC), within clinically identical tumors. Here, we show how activation of the Sonic Hedgehog (SHH) pathway in neural stem and progenitor cells creates a foundation for tumor cell evolution to heterogeneous states that are histologically indistinguishable but molecularly distinct. In spontaneous medulloblastomas that arise in Patched (Ptch)+/− mice, we identified three distinct tumor subtypes. Through cell type–specific activation of the SHH pathway in vivo, we determined that different cells of origin evolved in unique ways to generate these subtypes. Moreover, TICs in each subtype had distinct molecular and cellular phenotypes. At the bulk tumor level, the three tumor subtypes could be distinguished by a 465-gene signature and by differential activation levels of the ERK and AKT pathways. Notably, TICs from different subtypes were differentially sensitive to SHH or AKT pathway inhibitors, highlighting new mechanisms of resistance to targeted therapies. In summary, our results show how evolutionary processes act on distinct cells of origin to contribute to tumoral heterogeneity, at both bulk tumor and TIC levels. Cancer Res; 74(17); 4864–74. ©2014 AACR.

Expression patterns of the SHH pathway genes in spontaneous Ptch tumors.

SummaryThe stem cell theory that all blood cells are derived from hematopoietic stem cell (HSC) is a central dogma in hematology. However, various types of blood cells are already produced from hemogenic endothelial cells (HECs) before the first HSCs appear at embryonic day (E)11 in the mouse embryo. This early blood cell production from HECs, called HSC-independent hematopoiesis, includes primitive and definitive erythromyeloid progenitors that transiently support fetal blood homeostasis until HSC-derived hematopoiesis is established. Lymphoid potential has traditionally been detected in the extra-embryonic yolk sac (YS) and/or embryos before HSC emergence, but the actual presence of lymphoid progenitors at this stage remains unknown. In addition, whether HSCs in the fetal liver are the main source of innate-like B-1a cells has been controversial. Here, using complementary lineage tracing mouse models, we show that HSC-independent multipotent progenitors (MPPs) and HSC-independent adoptive B-lymphoid progenitors persist into adult life. Furthermore, HSCs minimally contribute to the peritoneal B-1a cell pool; most B-1a cells are originated directly from ECs in the YS and embryo and HSC-independent for life. Our discovery of extensive HSC-independent MPP and B-lymphoid progenitors in adults attests to the complex blood developmental dynamics through embryo to adult that underpin the immune system and challenges the paradigm of HSC theory in hematology.

Bladder cancer (BLCA) is the second most common urological malignancy in the US and is usually not curable at an advanced stage. The transcriptomic profiling data of bladder cancer revealed that 64% of BLCA patients have frequent dysregulation of the components of the SWI/SNF, a chromatin remodeling complex. Alterations in chromatin remodeling genes have been previously implicated in human oncogenesis. SMARCB1/INI1 is one of the critical core components of the SWI/SNF complex. In this study, we demonstrate that primary BLCA patients harboring SMARCB1 loss strongly associate with worse survival. Specifically, the biallelic inactivation of the SMARCB1 (SMARCB1 knockout (KO)) in BLCA cell lines resulted in the emergence of aggressive tumor growth leading to soft tissue metastasis to lungs, liver, kidney, and intestine in vivo. Mechanistic studies revealed that IL-6- STAT3 axis is upregulated in SMARCB1 KO BLCA cells. Our data suggest that the STAT3 specific inhibitor, inhibits invasive potential of SMARCB1 KO cells suggesting potential therapeutic efficacy for STAT3 inhibitor to treat subsets of BLCA harboring SMARCB1 loss. In addition, we identified a unique transcriptional signature activated upon SMARCB1 loss, which is predictive of poor patient outcome in BLCA, and hence may be clinically valuable. Bladder cancer (BLCA) is the second most common urological malignancy in the US and is usually not curable at an advanced stage. The transcriptomic profiling data of bladder cancer revealed that 64% of BLCA patients have frequent dysregulation of the components of the SWI/SNF, a chromatin remodeling complex. Alterations in chromatin remodeling genes have been previously implicated in human oncogenesis. SMARCB1/INI1 is one of the critical core components of the SWI/SNF complex. In this study, we demonstrate that primary BLCA patients harboring SMARCB1 loss strongly associate with worse survival. Specifically, the biallelic inactivation of the SMARCB1 (SMARCB1 knockout (KO)) in BLCA cell lines resulted in the emergence of aggressive tumor growth leading to soft tissue metastasis to lungs, liver, kidney, and intestine in vivo. Mechanistic studies revealed that IL-6- STAT3 axis is upregulated in SMARCB1 KO BLCA cells. Our data suggest that the STAT3 specific inhibitor, inhibits invasive potential of SMARCB1 KO cells suggesting potential therapeutic efficacy for STAT3 inhibitor to treat subsets of BLCA harboring SMARCB1 loss. In addition, we identified a unique transcriptional signature activated upon SMARCB1 loss, which is predictive of poor patient outcome in BLCA, and hence may be clinically valuable. Citation Format: Chandra Sekhar Amara, Karthik Reddy Reddy, Yuntao Yang, Badrajee Waduge Danthasinghe, Allison Bellman, Andrea B. Apollo, David J. Shih, Leomar Ballester, Pavlos Msaouel, Wenjin J. Zheng, Matthew J. Ellis, Seth P. Lerner, Arun Sreekumar, Shyam M. Kavuri, Nagireddy Putluri. Elucidating the role of SMARCB1 in bladder cancer progression and metastasis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2417.

We retrospectively evaluated 233 incident patients (61% black, 27% white, and 12% Hispanic/Asian) to our peritoneal dialysis (PD) program from January 1987 to September 1997 to identify any possible racial differences in patient survival. Information collected included clinical features, comorbid conditions, nutritional status, and dialysis dose at initiation of dialysis. The average age was 52 ± 16 (SD) years, and 49% were men. Diabetes mellitus was present in 41% of patients. Overall follow-up was 31 ± 24 (median 26) months during which time 21% of patients underwent transplant, 29% of patients transferred to hemodialysis (HD), and 42% of patients died. The Cox proportional hazards analysis, based on intent-to-treat, identified age (RR: 1.03), race (RR: 2.35, white versus black), cardiac disease (RR: 1.97), and serum albumin (RR: 0.44) to independently predict mortality. Further analysis was performed based on diabetic status, and the analysis identified age (RR: 1.06), race (RR: 2.45, white versus black), and peripheral vascular disease (RR: 2.88) as predictors of mortality in diabetic patients. In nondiabetic patients, age (RR: 1.03), race (RR: 2.24, white versus black), cardiac disease (RR: 2.48), cerebrovascular disease (RR: 3.17), and serum albumin (RR: 0.39) were significant predictors of mortality. The significance of race persisted even after adjusting patients transferring to hemodialysis. The adjusted patient survival at 1, 2, and 5 years was 94%, 87% and 53% for black patients, and 86%, 72%, and 23% for white patients. The adjusted patient survival in diabetics at 1, 2, and 5 years was 92%, 79%, and 37% for black patients, and 82%, 56%, and 9% for white patients. The adjusted patient survival in nondiabetics at 1, 2, and 5 years was 94%, 91%, and 63% for black patients, and 88%, 82%, and 35% for white patients. In conclusion, long-term patient survival is better for black patients than white patients in our peritoneal dialysis program. Peritoneal dialysis should be considered a viable dialytic option for black patients entering an end-stage renal disease program.

Objective To heighten the awareness of a possible association of sclerosing peritonitis in patients with systemic lupus erythematosus (SLE). Methods and Results Over the course of 17 years (from January 1981 to December 1997), 371 patients were treated with continuous ambulatory peritoneal dialysis (CAPD) at Rush–Presbyterian–St Lukes Medical Center. The patients were followed on CAPD for an average of 25 ± 21 (SD) months with a median of 19 months (range 0.2 – 115 months). During this time only 2 (0.5%) patients were diagnosed with sclerosing peritonitis, and both had SLE with ongoing evidence of active disease while on CAPD. With a total of 26 SLE patients being treated with CAPD during the observation period, the prevalence of sclerosing peritonitis can be said to be as high as 8% in this patient population. Conclusion These cases suggest that autoimmune diseases, such as SLE, that are well known to cause immune-mediated serositis may represent an additional factor predisposing to the development of sclerosing peritonitis in patients treated with CAPD.

We report two patients treated with ciprofloxacin who presented with acute renal failure. On renal biopsy, a necrotizing vasculitis was identified in addition to acute interstitial nephritis. Improvement in renal function resulted with the discontinuation of the antibiotic and the institution of immunosuppressive therapy.