2019 — 2021 |
Puram, Sidharth Venkata |
K08Activity Code Description: To provide the opportunity for promising medical scientists with demonstrated aptitude to develop into independent investigators, or for faculty members to pursue research aspects of categorical areas applicable to the awarding unit, and aid in filling the academic faculty gap in these shortage areas within health profession's institutions of the country. |
Understanding Programs of Invasion and Metastasis in Head and Neck Cancer
PROJECT SUMMARY Intra-tumoral heterogeneity in human cancers is associated with poor treatment response and prognosis. Head and neck squamous cell carcinoma (HNSCC) is a strikingly heterogeneous tumor, with over 550,000 new cases per year. Unfortunately, this heterogeneity represents a major challenge in targeted therapy and treatment responses, with detailed maps of intra-tumoral heterogeneity just starting to be defined. During my Ph.D. training with Dr. Azad Bonni, I studied normal processes of brain development using sophisticated biochemistry techniques1,2,3,4, which inspired me to understand the signaling pathways that drive specific biological programs. I applied these skills to studies of glioma, ultimately, defining a dual role for the transcription factor STAT3 as a tumor suppressor and an oncogene5,6,7. For my post-doctoral training, I joined the laboratory of Dr. Bradley Bernstein, an expert in transcription, epigenetics, and innovative technology, to combine my interests in tumor heterogeneity with my clinical interests in head and neck surgical oncology. Using single cell RNA-seq, we defined the first atlas of HNSCC, identifying a partial epithelial-to-mesenchymal (p-EMT) program associated with lymph node metastasis, lymphovascular invasion, and extracapsular extension8. We also refined tumor subtyping from bulk RNA-seq approaches such as the Cancer Genome Atlas by leveraging our single cell profiles. The objectives of this proposal are to (1) determine expression heterogeneity within 2D and 3D head and neck cancer models and identify suitable models of p-EMT, (2) investigate the regulatory influence of cancer- associated fibroblasts and their ligands on malignant cancer cell states, including the underlying transcriptional and epigenetic mechanisms, and (3) define the signaling pathways downstream of p-EMT and discern their functional importance in tumor behaviors. Dr. Tim Ley and Dr. Greg Longmore are exceptional mentors with an extensive history of trainees in major academic positions. Dr. Ley is an internationally renowned leader in oncology, genomics, and tumor biology, with appointments including Associate Director of the Siteman Cancer and the McDonnell Genome Institute at Washington University St. Louis (WUSTL), National Cancer Advisory Board Member, and former Chair of Board of Scientific Counselors for National Institute Genome Research Institute. Similarly, Dr. Greg Longmore is a world expert in molecular oncology, EMT, and cell biology, with appointments as Co-Director of Molecular Oncology and Director of the Integrating Communications within the Cancer Environment Institute, WUSTL. An exceptional Research Advisory Committee (RAC) will provide additional guidance, including Dr. Ting Wang, Dr. Jose Zevallos, and Dr. Rob Mitra. The K08 award will provide me with the ideal opportunity to succeed in my career goals. The detailed training plan includes a RAC and development of technical/leadership skills that will be invaluable for a transition to independence. Research will be carried out at WUSTL, a prestigious research institute that fosters collaboration, emphasizes intellectual exchange, and boasts unparalleled facilities. 1. Kim AH, Puram SV, Bilimoria PM et al. Cell 136(2): 322-336 (2009). 2. Puram SV, Kim AH, Ikeuchi Y, et al. Nature Neuroscience 14(8): 973-983 (2011). 3. Puram SV, Riccio A, Koirala S et al. Genes and Development 25(24): 2659-2673 (2011). 4. Puram SV*, Kim AH*, Park HY et al., Cell Reports 4(1): 19-30 (2013). 5. de la Iglesia N, Konopka G, Puram SV et al., Genes and Development 22(4): 449-462 (2008). 6. Puram SV, Yeung CM, Asl-Jahani A, et al. Journal of Neuroscience 32(23): 7806-7818 (2012). 7. Jahani-Asl A, Yin H, Soleimani VD et al., Nature Neuroscience 19(6): 798-806 (2016) 8. Puram SV*, Tirosh I*, Parikh A* et al., Cell 171(7): 1611-1624 (2017)
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0.965 |
2021 |
Puram, Sidharth Venkata |
R21Activity Code Description: To encourage the development of new research activities in categorical program areas. (Support generally is restricted in level of support and in time.) |
Rna Modification and the Regulation of Partial Emt in Head and Neck Cancer
PROJECT SUMMARY Head and neck squamous cell carcinoma (HNSCC) is the sixth leading cause of cancer-related mortality, with the majority of deaths attributable to tumor metastasis and failures in treatment. Because most cases of HNSCC result from tobacco and alcohol exposure, these tumors are highly heterogeneous, greatly complicating diagnosis, treatment, and investigations into the biology of this disease. We recently performed single cell RNA- sequencing (scRNA-seq) in HNSCC and identified a partial epithelial-to-mesenchymal (p-EMT) transcriptional program that is predictive of poor clinical outcomes including nodal metastasis and diminished survival (Puram et al., Cell). Understanding the regulatory factors that control the p-EMT program in HNSCC is of critical importance as targeting multiple genes in a complex pathway such as p-EMT is particularly challenging, yet has the potential to significantly improve HNSCC outcomes and treatment decision-making. RNA modification proteins, which can directly read/write nucleotide marks on RNA, have emerged as one exciting class of such regulatory proteins. Because these proteins modulate multiple RNA transcripts, targeting RNA modification proteins may disrupt expression of multiple disease-related genes and make resistance less likely to emerge. In HNSCC, we have found that METTL3, an RNA methyltransferase which catalyzes the N6-methyladenosine (m6A) modification, is a key regulator of the p-EMT program in HNSCC. In preliminary studies, we have discovered that perturbation of METTL3 in HNSCC disrupts p-EMT signaling and reduces invasion in vitro. However, the precise mechanisms by which METTL3 and other RNA modification proteins exert their function are poorly understood, but may depend on changes in transcript stability and/or translation. Thus, a better understanding of how METTL3 modulates p-EMT in HNSCC is likely to improve rational drug design and future small molecule- and biologically-based screens in search of effective epitranscriptomic therapeutics. We hypothesize that METTL3 antagonizes p-EMT in HNSCC by disrupting the translation of critical p-EMT target genes. To test which domains in METTL3 are essential for its function, we will first perform sophisticated structure-function analyses in HNSCC cell lines and patient-derived xenograft organoid (PDXOs) (Aim 1). We will determine which domains are required for m6A function using state-of-the-art mass spectrometry methods, while also mapping the binding partners of the critical domains. To determine if METTL3 controls the transcription or translation (or both) of p-EMT genes, we will utilize advanced biochemical techniques including meRIP-seq to study the stability of p-EMT RNA transcripts and polysome profiling and PAR-CLIP to investigate the effects of METTL3 on translation of p-EMT genes (Aim 2). These studies will provide indispensable insight into the mechanism by which METTL3 directs HNSCC tumorigenesis, specifically focusing on its regulation of p-EMT signaling, and thereby revealing METTL3 domains that could be targeted by new therapeutics to more effectively and specifically treat HNSCC.
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0.965 |