Cellular and Molecular Mechanisms of Early onset colorectal cancer

Since 1990, the overall incidence of late onset colorectal cancer (LOCRC ≥50 yrs. old) has decreased. However, these rates have steadily increased in younger individuals (<50 yrs. old). The cellular and molecular mechanisms underlying early onset CRC (EOCRC), and its poorer survival outcomes in men are unclear. Approximately 80% of EOCRC tumors are sporadic, arising from acquired genetic mutations. Epidemiological studies have noted a higher incidence and advanced-stage cancers in males with EOCRC compared to females, with males exhibiting a larger Wnt/β-catenin signaling component, a major driver of cancer stem cell (CSC) population expansion. The enzyme asparagine synthetase (ASNS) links CSC function and sex differences to EOCRC development. Targeting ASNS-expressing ISCs could offer therapeutic potential as high tumoral asparagine (Asn) levels correlate with cancer aggressiveness in male EOCRC alone. The objective of this proposal is to investigate sex- dependent mechanisms in EOCRC progression. My central hypothesis is that ASNS drives CSC expansion and tumorigenesis in EOCRC, which is enhanced in males. I will test my hypothesis through the following Specific Aims. Aim 1 (K99 phase): Analyze untargeted metabolomics data of 372 paired CRC and normal mucosa tissues from EOCRC and LOCRC. I will perform whole exome sequencing and use machine learning to identify unique mutational signatures and metabolites associated with EOCRC. Aim 2 (K99 phase): Develop patient- derived organoids (PDOs) from EOCRC/LOCRC samples, supplement them with Asn at varying concentrations, and examine organoid number, growth, size, and crypt budding. I will assess CSC dynamics in PDOs using time- of-flight mass cytometry (CyTOF). I will delete ASNS in PDOs using CRISPR-Cas9 and test the sex-specific effects of ASNS loss on PDO growth and crypt budding. Aim 3 (R00 phase): Evaluate the effects of ASNS deletion on tumor growth and survival using a patient-derived xenograft model. Investigate tumor heterogeneity using single cell/nuclei RNA-Sequencing and spatial transcriptomics. This research will provide novel insights into the sex-dependent roles of ASNS and Asn in ISC niche expansion during EOCRC progression, improving our understanding of EOCRC development. The training will equip me with skills in machine learning, stem cell biology, and tumor heterogeneity, which will be foundational for developing pre-clinical models of EOCRC in my future independent laboratory. Project Number: 1K99CA304369-01A1 | Fiscal Year: 2026 | NIH Institute/Center: National Cancer Institute (NCI) | Principal Investigator: Oladimeji Aladelokun | Institution: YALE UNIVERSITY, NEW HAVEN, CT | Award Amount: $130,490 | Activity Code: K99 | Study Section: Special Emphasis Panel[ZRG1 CDPT-N (55)] View on NIH RePORTER: https://reporter.nih.gov/project-details/11357472