Origin licensing and MCM complex regulation in human lymphocytes

The National Institute of Allergy and Infectious Diseases awarded The Trustees of Columbia University in the City of New York $100,228 on November 23, 2026, under the Allergy and Infectious Diseases Research program (CFDA 93.855).

The award funds research into origin licensing and MCM complex regulation in human lymphocytes, specifically investigating lineage-specific differences in how natural killer cells and CD8+ memory T cells regulate cell cycle entry and respond to replication stress. Natural killer cell deficiency is a rare primary immunodeficiency disorder linked to partial loss-of-function variants in MCM4 genes, which are core components of the DNA helicase complex responsible for unwinding DNA during genome replication. The research examines why natural killer cells are uniquely sensitive to replication stress compared to CD8+ memory T cells, despite both cell types sharing cytotoxic functions. Understanding these mechanistic differences may inform development of therapies for individuals with MCM-linked NK cell deficiency who experience moderate to severe viral infections and increased cancer susceptibility.

The recipient is Columbia University's Health Sciences Division. Performance occurs in New York, New York, with a period of performance ending August 31, 2028.

Abstract

Natural killer (NK) cells are innate immune cells that recognize and kill virally infected and cancerous cells. NK cell deficiency is a rare primary immunodeficiency disorder characterized by impaired NK cell development or function that leads to moderate to severe viral infections and increased cancer susceptibility. While only a handful of genes have been identified that cause isolated NK cell deficiency, these include genes in the CDC45-MCM-gins (CMG) complex. This complex is the core enzymatic subunit of DNA helicase responsible for unwinding DNA at replication forks during genome duplication. Adequate loading of the MCM complex onto DNA in G1, known as origin licensing, ensures faithful duplication of the genome. Aberrant MCM2-7 loading or partial loss of function helicase variants increase replication fork stalling, DNA breaks, and chromosomal instability. To safeguard DNA synthesis, the origin licensing checkpoint may be activated to slow down G1 progression and ensure adequate MCM complex loading. Partial loss of function variants in MCM4 cause NK cell deficiency with genome instability, cell cycle arrest, and impaired proliferation. NK cells are also more sensitive to replication stress than t cells, as evidenced by their increased DNA damage and apoptosis in response to mild replication stress. The cellular mechanism underlying NK cell-specific sensitivity to helicase variants remains a mystery and highlights lineage-specific differences in how lymphocytes regulate cell cycle entry. To address this knowledge GAP, i aim to understand the unique cell cycle kinetics and origin licensing checkpoint usage in human NK cells and CD8+ memory t cells, which share cytotoxic functions with NK cells, but are unaffected in NK cell deficiency. I hypothesize that differential loading of the MCM complex and usage of the origin licensing checkpoint under differential activation conditions in NK cells and CD8+ t cells contribute to the sensitivity of NK cells to mutations in the MCM complex. In aim 1, i will investigate the usage of origin licensing checkpoints in mature activated NK cells and t cells by characterizing the loading of the MCM complex onto DNA and slowing down origin licensing. This will be achieved using RT-qpcr, chromatin flow cytometry, single-chain antibody auxin-inducible degron 2 system, and high-resolution confocal microscopy. In aim 2, i will investigate the impact of MCM complex expression signal strength on NK cells and t cells by examining lineage-specific activation-induced signaling of MCM complex proteins through RT-qpcr, phospho- flow cytometry, and car t cell receptor knock-in. By completing these aims, i will uncover critical lineage- specific mechanisms that govern cell cycle entry in human immune cells and explain how disruptions in these processes contribute to immunodeficiency and heightened risk of disease.