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High throughput screening to generate and investigate cancer driver mutations in hepatocellular carcinoma.

Award Number: F30CA306325
ORGANIZATION: NATIONAL CANCER INSTITUTE
OPDIV: NIH
AWARD CLASS: DISCRETIONARY
AWARD ACTIVITY TYPE: FELLOWSHIP/SCHOLARSHIP/STUDENT LOANS
PERIOD OF PERFORMANCE START DATE: 02/15/2027
PERIOD OF PERFORMANCE END DATE: 02/14/2028

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High throughput screening to generate and investigate cancer driver mutations in hepatocellular carcinoma. - Project Summary Cancer remains a leading cause of death in the United States and globally. Understanding the genetic mutations that drive cancer can help inform diagnosis, prognosis, and treatment options. Cancer driver mutations are thought to cause tumor formation and malignancy by leading to abnormal and uncontrolled cellular growth. To date, thousands of cancer driver mutations have been identified in hundreds of genes. Experimental models that precisely install cancer driver mutations are essential for investigating the mechanisms that underlie cancer. CRISPR-based prime editing offers a comprehensive way to generate a diverse array of cancer driver mutations, including substitutions, insertions, deletions, or combinations. In prime editing, a prime editing guide RNA (pegRNA) guides mutant Cas9 to nick one strand of the genome target site and serves as a template for reverse transcriptase (fused to Cas9) to copy new information into the genome. However, designing and validating effective pegRNAs is expensive and time-consuming. Preliminary experiments using a high- throughput screening approach based on Next Generation Sequencing identified pegRNAs that guide efficient editing. This screen enabled simultaneous testing of thousands of pegRNA designs for their ability to edit frequent mutations in hepatocellular carcinoma (HCC), the most prevalent form of primary liver cancer. This project aims to use these pegRNA constructs to further characterize HCC mutations and to generate a mouse model of HCC. Aim 1 will characterize the cellular and molecular effects of HCC driver mutations. Utilizing a library of the highest efficacy pegRNAs from the preliminary screen, a secondary screen will be performed to identify mutations that confer a growth advantage in transformed primary human hepatocyte cell lines. Cellular fitness results from this screen will be validated using a competition assay. Cellular and molecular phenotypes of mutations that promote fitness will then be characterized. Colony formation assays will determine the ability of cells with cancer driver mutations to expand into a clonal population. RNA sequencing will reveal transcriptional profiles associated with the cancer mutation. Aim 2 will model HCC driver mutations in vivo. Prime editing will be used to install cancer mutations in transformed primary human hepatocytes, and cells will be orthotopically implanted into the livers of immune-deficient mice via intrahepatic injection. After a period of growth, tumor formation and the histopathology and gene expression signatures of the tumors will be analyzed. This study will be piloted with pegRNAs (identified from the preliminary screen) that generated an activating mutation in the conserved gene NOTCH1. In vivo models of cancer driver mutations will be used to assess how tumors respond to treatment with first-line therapies for HCC. Successful completion of this project will generate a system to characterize HCC driver mutations at the cellular level and in tumors, informing potential treatment approaches based on the genetic profiles of cancer.


Issue Date FY	Funding FY	Legal Entity Name	Legal Entity Address	Legal Entity City	Legal Entity State	Legal Entity Zip Code	Legal Entity COUNTY	Legal Entity COUNTRY	Assistance Listing	Award Code	Budget Year	Action Date	Action Type	Action Amount

Issue Date FY: 2026 ( Subtotal = $36,081 )
2026	2026	UNIVERSITY OF MASSACHUSETTS MEDICAL SCHOOL	55 LAKE AVE N	WORCESTER	MA	01655	WORCESTER	USA	Cancer Research Manpower	000	1	8/17/2026	NEW	$36,081
														Subtotal = $36,081

Grand Total All Awards = $36,081

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High throughput screening to generate and investigate cancer driver mutations in hepatocellular carcinoma.

Award Number: F30CA306325

ORGANIZATION: NATIONAL CANCER INSTITUTE

OPDIV: NIH

AWARD CLASS: DISCRETIONARY

AWARD ACTIVITY TYPE: FELLOWSHIP/SCHOLARSHIP/STUDENT LOANS

PERIOD OF PERFORMANCE START DATE: 02/15/2027

PERIOD OF PERFORMANCE END DATE: 02/14/2028

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