United States

In our previous work, we identified clustering of kidney cancers based on differential metabolic pathway activation, and an association between metabolic clustering and survival in renal cell carcinoma. Here we further study clear cell renal carcinoma, papillary cell renal carcinoma and chromophobe renal cell carcinoma by applying machine learning models to pinpoint specific metabolic reactions, and their master regulators, that best explain differences in survival in these cancers. We constructed a cohort of 941 patients in The Cancer Genome Atlas (TCGA) diagnosed with kidney cancer (project codes KIPR [papillary cell RCC], KIRC [clear cell RCC] and KICH [chromophobe RCC]). We used tumor RNA-Seq data to construct reaction activity scores (RAS) using metabolic genes according to the HMRcore rules and taking into account enzymatic subunits and multiple different genes coding for proteins that catalyze the same reaction. We then used a machine learning model to estimate survival as a function of metabolic pathways, age and gender. By using Cox regularization with elastic-net penalty we were able to simultaneously perform variable selection and estimation of survival in presence of a large number of regressors. Using master regulatory analysis, we estimated activity of key transcription factors and then used a multinomial model with elastic net penalty to identify those key transcription factors that have the greatest impact on dysregulation of tumor metabolic pathways in renal cell carcinomas. Using glucose transport as a known hallmark of renal cell carcinoma, we identified 19 metabolic reactions that are better predictors of survival than glucose transport in our machine learning model. The metabolic reactions we identified fit into larger categories that include folate and protein metabolism, as well as beta-oxidation. These are consistent with metabolic changes induced by mTOR, and it is notable that inhibitors of mTOR are already used in the clinic for treatment of renal cell carcinoma. Our research raises the question of whether effectiveness of mTOR inhibitors can be increased by amplifying the effects of mTOR inhibition on oncogenesis through targeting of related master regulators of metabolism.

AMA Research Challenge 2022

Analysis of Metabolic Profile and Survival Data in Renal Cancer

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Ricardo Correa, MD, EdD
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Jessica Li, MD/PhD Candidate
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Charles Lopresto, DO

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Welcome to the largest national, multi-specialty research event featuring research from 750 medical students, residents and fellows, and international medical graduates.


poster

Multiple myeloma (MM) is a plasma cell malignancy that grows in the bone marrow and is characterized by severe bone destruction. The growth of MM cancer cells in the bone marrow increases osteoclasts, bone-resorbing cells, which create debilitating bone lesions. MM cells also decrease osteoblasts, bone-forming cells, which reduces bone formation. Despite recent advances in anti-MM therapy, MM patients frequently relapse and exhibit high fracture incidence even during disease remission. Our lab showed that inhibition of Notch, a pathway regulating cell-to-cell communication in the tumor niche, with a bone-targeted Notch inhibitor (BT-GSI) decreases tumor growth and bone resorption without inducing toxicity. We also found that pharmacologic inhibition of Sclerostin (Scl-Ab), a potent inhibitor of Wnt signaling and osteoblast function, increases bone formation without affecting tumor growth. In this study, we hypothesized that co-administration of BT-GSI and Scl-Ab simultaneously decreases tumor burden and improves bone health by inhibiting bone resorption and increasing bone formation. 6-week-old immunocompetent mice were injected intratibially with 105 murine 5TGM1 myeloma cells. After four weeks, mice with MM were randomized to groups receiving vehicle, Scl-ab (100mg/Kg/1x/wk), BT-GSI (2.5mg/Kg/3x/wk), or Scl-ab+BT-GSI combined for four additional weeks. Longitudinal micro-CT was performed every four weeks to assess bone mass and microarchitecture. Alizarin Red and Calcein fluorescent dyes were injected 5 and 2 days, respectively, before sacrifice. Bone dynamic and static histomorphometry analyzed fluorescent labels and osteoclasts present via TRAP staining using OsteoMeasure. Mice treated with Scl-Ab therapy exhibited similar tumor burden levels than mice receiving vehicle injections. In contrast, mice treated with BT-GSI, either alone or in combination with Scl-ab, exhibited decreased tumor burden compared to vehicle-treated mice. Mice receiving vehicle injections had decreased bone mass compared to control naive mice. All three therapies significantly increased bone mass compared to vehicle mice. There was no organ toxicity with any of the therapies. We performed static and dynamic histomorphometric analysis to determine the mechanism underlying the skeletal protective effects. Vehicle-treated mice had decreased bone formation rate (BFR) compared to naïve mice, although not statistically significant. Scl-ab therapy increased BFR by 28%, bringing it back to naïve levels. BT-GSI therapy did not affect BFR, which remained decreased compared to naïve mice. Co-administration of BT-GSI and Scl-ab also restored BFR to naive mice levels. Vehicle-treated mice had a five-fold increase in osteoclast number corrected by bone surface (Oc.N/BS) compared to naïve mice. Scl-ab and BT-GSI induced a 39% and 75% decrease in Oc.N/BS compared to MM-bearing mice receiving vehicle, respectively. Co-administration of BT-GSI and Scl-ab also resulted in a 75% reduction in Oc.N/BS compared to MM mice, bringing Oc.N/BS back to levels found in naïve mice. BT-GSI + Scl-ab combination therapy decreases tumor burden and improves bone health by decreasing osteoclasts and increasing the bone formation rate. BT-GSI + Scl-ab combination therapy is more effective than these therapies alone in stopping tumors and repairing damaged bone. Because this bone-targeted multimodal therapy lacks toxicity, it provides a new therapeutic avenue to control MM growth and improve bone health in MM patients.

Bone-targeted combination therapy decreases tumor progression and restores bone mass by increasing bone formation in mice with established multiple myeloma

Lung cancer is the leading cause of cancer-related death in both men and women in the United States. Non-small cell lung cancer is the most prevalent and includes adenocarcinoma (AD), and squamous cell lung cancers. The unique cellular biology of these cancers could potentially allow for serological detection and identification via tissue specific protein markers. Galectins are a group of β-galactoside binding proteins which are known to be alternatively expressed in cancers and are being studied for their biomarker potential. Galectin-7 is expressed in epithelial tissues as well as in all layers of the epidermis as it is normally highly expressed by squamous cells. We evaluated the potential for galectins to be used as a serum biomarkers and delineators of two non- small cell lung cancer subtypes.
The serum concentrations of galectin-7 were measured in 85 lung cancer patients using ELISA. Forty-eight samples were from adenocarcinoma patients and 37 were from squamous cell carcinomas patients. The serum galectin-7 concentrations in cancer patients were compared to healthy controls and by their histological subtypes. Oneway analysis was performed using Tukey-Kramer HSD for pairwise comparisons.
Galectin-7 concentrations were significantly elevated in serum samples from squamous cell lung cancer patients (mean, 2.45 ng/mL) relative to healthy controls (N, 26; mean, 1.16 ng/mL; p- value, 0.0010) and relative to adenocarcinoma lung cancer patients (mean, 1.47 ng/mL; p-value, 0.0040).
These results show that serum galectin-7 is increased in patients with squamous cell lung cancer compared to adenocarcinoma lung cancer. Further benchwork would include (1) measuring serum concentrations of galectin-7 by stage of squamous cell lung cancer, (2) identifying concentrations of serum galectin-7 in other lung cancer subtypes, and (3) in other non-pulmonary squamous cell carcinomas such as skin, cervical, and head and neck. Potential clinical applications of this work would include the measurement of galectin-7 in blood samples of cancer patients to aid in cancer screening and tumor type identification.

Comparison of Galectin-7 Serum Concentrations in Squamous Cell and Adenocarcinoma Lung Cancers

Concurrent malignant and benign nephrosclerosis in an elderly cadaver with previously diagnosed cerebral atherosclerosis

Background: 4-1BBL is a co-stimulatory molecule that is important for T-cell expansion and
induction of robust CD8+ T-effector and memory responses, critical for tumor rejection. The natural 4-1BBL is a trimeric transmembrane protein that has no activity in soluble form. To overcome this limitation, a unique soluble form of 4-1BBL was developed by linking core streptavidin (SA) to the extracellular domain of murine 4-1BBL (SA-4-1BBL). This chimeric protein forms oligomers that have pleiotropic effects on cells of innate, adaptive, and regulatory immunity. However, production and purification of SA-4-1BBL is cost- and time-intensive. Therefore, development of an expression platform that is more efficient, translational, and augments anti-tumor efficacy of SA-4-1BBL will have a significant impact for clinical development of this immune adjuvant. Thus, we developed a novel oncolytic adenoviral system to deliver SA-4-1BBL to solid tumors.
Methods: The AdenoQuick system was used to construct OAds expressing SA-4-1BBL or SA (control). Transgene expression was confirmed by western blot and SA-4-1BBL biological function was evaluated by splenocyte proliferation assay in vitro. The OAdSA-4-1BBL and
Ó 2022 American Medical Association. All rights reserved.
OAdSA-mediated cancer killing effect was evaluated by crystal violet staining, the activation of autophagy-mediated immunogenic cell death was assessed by detection of autophagy markers LC3-I, LC3-II and p62. The damage-associated molecular patterns molecules adenosine triphosphate, high-mobility group box 1, and calreticulin were evaluated by luminescence, ELISA, and flow cytometry, respectively. Tumor expression of SA, SA-4-1BBL, and Hexon was evaluated by immunohistochemistry. The OAdSA-4-1BBL therapeutic efficacy was evaluated in C57BL/6 mice-bearing subcutaneous TC-1 lung tumor.
Results: We successfully constructed OAds expressing SA or SA-4-1BBL. We demonstrated that SA-4-1BBL can produce biologically active oligomers, selectively induces autophagy- mediated immunogenic cell death in murine and human lung cancer cell lines with little cytotoxicity to normal cell lines. The transgene expressions within the subcutaneous tumor were confirmed and OAdSA-4-1BBL shows greater anti-tumor efficacy than control viruses in a TC-1 tumor mouse model.
Conclusion: Overall, our data suggests that OAd expressing the immune checkpoint stimulator SA-4-1BBL may represent alternative therapeutic strategy against lung cancer, which could be used alone or in combination with immune checkpoint inhibitors, or other approaches aimed at enhancing the anti-tumor immune response.

Development and characterization of an oncolytic adenovirus armed with the immune checkpoint stimulator SA-4-1BBL

Effect of Hesperetin against DMH induced toxicity in the colon of Wistar rats

Background
Glioblastoma (GBM) represents the most common primary malignant brain tumor in adults, with a median survival rate of less than two years. GBM presents with therapeutic resistance and high rates of recurrence, largely attributed to the presence of glioblastoma stem cells (GSCs). GSCs are frequently located in specific niches, including a hypoxic niche and a normoxic, perivascular niche. Targeting GSCs is a potential therapeutic avenue for treating GBM, but the details of GSC function in hypoxic conditions remains poorly understood. Further, most gene expression findings are limited to epigenetic or transcriptional regulation assays, overlooking the importance of the translatome. This study expands on conclusions made using steady-state mRNA levels by including ribosome profiling to evaluate translational regulation mechanisms. 

Methods
We selected two patient-derived GSC lines (GSC 215, GSC 316) and cultured them in both hypoxic (5% O2) and normoxic (21% O2) conditions. RNA-seq libraries were prepared in parallel with RIBO-seq libraries for the lines in both conditions, with biological replicates for each treatment group, after which Illumina sequencing was performed. Raw sequencing reads were assessed for quality. Single-end reads were aligned and counted using command line versions of STAR, BEDTools, and SAMTools. Differential changes in ribosome-protected fragments (∆RPF) and changes in transcript levels (∆RNA) were calculated using DESeq2. GO term analyses for transcripts was performed with Reactome using a hypergeometric statistical test and BH FDR correction.

Results
Comparing ∆RNA and ∆RPF levels between hypoxic and normoxic GSCs reveals 9 functional clusters of genes. These categories indicate genes which are post-transcriptionally controlled in 3 manners: translationally suppressed (28.19%), translationally upregulated (32.87%), or concurrently translated (38.93%). Altogether, more than 60% of mRNA show marked regulation on the translational level.

Significant enrichment of genes in the NOTCH signaling pathway (p = 1.09e-02), WNT signaling pathway (p = 1.37e-04), and RHO GTPase cycle (p = 2.37e-07) are present among the transcripts that experience translational upregulation in hypoxic conditions.

Conclusion
This study may lead to the identification of potential therapeutic targets as evidenced by their function as regulatory hubs in cellular networks responsible for hypoxic adaptation. These observations are largely undetected by RNA-seq measurements.


Evaluating the Role of Translation Regulation in Glioblastoma Stem Cells for Hypoxic Adaptation

First-in-class Heterobifunctional Proteomimetic Polymer Capable of Direct Inhibition and Targeted Degradation of Myc

In silico modeling of LAT1 efflux agonists for the treatment of pancreatic cancer

Neuroblastoma is the most common extracranial solid tumor in children. It accounts for more than 7% of malignancies in patients less than fifteen years of age and makes up 15% of oncology deaths. Neuroblastoma is a cancer derived from primordial neural crest cells under conditions of inappropriate migration, maturation, and or differentiation. These extracranial tumors are formed in the adrenal medulla, sympathetic ganglia, and abdomen. Current treatments for neuroblastoma include surgery, chemotherapy, radiotherapy, and biotherapy. Interestingly, neuroblastoma tumors are heterogenous with some children having low-risk tumors that regress entirely while others have high-risk tumors with widespread metastasis and poor outcomes. The expression of MYCN, a gene in a family of oncogenic drivers, in neuroblastoma tumors has been associated with high-risk neuroblastoma and worse outcomes. Children with high-risk neuroblastoma account for approximately half of all patients diagnosed with neuroblastoma. Despite current interventions and aggressive treatment, children with high-risk neuroblastoma have a long-term survival rate of less than 50%. Currently, there are not many targeted therapies for high-risk neuroblastoma as there is little know about the mechanism driving this cancer. By identifying the mechanism driving tumor progression, a treatment to specifically target the different steps of this cancer can be created. Signaling through neuronal nicotinic acetylcholine receptors (nAChRs) is involved in many neuronal processes of neural tissue, including the proliferation of neural crest cells. These receptors are a diverse group of ligand-gated pentametric channels can exist as homopentamers or heteropentamers of α or β subunits. The only mammalian homopentamer is the α7 receptor that when activated is suggested to promote neural cell survival. Furthermore, expression of nAChR has been shown to be regulated by MYCN. Our lab is interested to see if MYCN-expressing neuroblastoma tumors upregulate the expression of nAChRs leading to an overall drive in tumor progression. Therefore, targeting this interaction between nAChRs and MYCN can become a potential therapy of high-risk neuroblastoma. 


N-MYC Transcription Factor Increases the Expression of Alpha-7 Nicotinic Acetylcholine Receptor Promoting Cell Viability in Neuroblastoma Cell Line

Background and Premise: Triple-negative breast cancer (TNBC) is an aggressive, metastatic disease with high mortality. The standard-of-care neoadjuvant chemotherapy (NACT) shows an initial response but followed by a drug resistance, increase in tumor relapse and distant metastases. Immunotherapy against programmed death ligand-1 (PD-L1) has shown efficacy in treating a subset of TNBC patients. However, it shows resistance in other patients due to new immune checkpoints such as VISTA, TIGIT, and LAG3. 

eIF4A1 is a mRNA helicase known to translate many oncogenic mRNAs including survivin, c-MYC, ROCK1, STAT1, ARF6 etc., We previously established that eIF4A1 is a vulnerable target in drug-resistant TNBC. Interestingly, we observed a reduction in levels of PD-L1 when eIF4A1 is targeted. Many downstream effectors of eIF4A1 such as c-MYC, STAT1 and ARF6 are indeed upstream regulators of PD-L1. This premise formed the basis to co-target eIF4A1 and PD-L1. Before that, we need to establish the clinical relevance of these targets in human TNBC biospecimens. This will ascertain the differential operation of the eIF4A1-PD-L1 axis in therapy-naïve and drug-resistant primary and mTNBC.

Approach: Human biospecimens obtained from CHTN NIH repositories (more than 100 TNBC primary tumor and metastatic biospecimens) will be analyzed for role of eIF4A1 in translation of oncogenic mRNAs that contribute to PD-L1 and other emerging immune checkpoint markers such as VISTA, TIGIT, and LAG3 via immunohistochemistry (after deglycosylation for effective detection of PD-L1) and also by immunoblotting. 

Results: The levels of total eIF4A1 from 16 matched, therapy naive TNBC tissue were compared to adjacent normal breast tissue from the same patient via immunoblotting. Total eIF4A1 levels were found to be significantly elevated in TNBC compared to adjacent normal tissue. The downstream effectors of eIF4A1 such as STAT1 and ARF6 were also significantly elevated indicating a robust helicase activity of eIF4A1. Importantly, PD-L1 levels was found to be elevated. Additionally, the total eIF4A1 level were elevated in drug-resistant tumors than normal breast tissue samples as assesses by immunohistochemistry. 

Conclusion: The eIF4A1-STAT1-PD-L1 axis is highly active in TNBC especially in drug-resistant situations. Currently, we are evaluating other immune checkpoints such as VISTA, TIGIT, and LAG3. 


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Analysis of Metabolic Profile and Survival Data in Renal Cancer

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Bone-targeted combination therapy decreases tumor progression and restores bone mass by increasing bone formation in mice with established multiple myeloma

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