# The role of DNA-PKcs in DNA repair, lymphocyte development, RNA metabolism and tumor suppression

> **NIH NIH R01** · COLUMBIA UNIVERSITY HEALTH SCIENCES · 2024 · $471,356

## Abstract

PROJECT SUMMARY/ABSTRACT
 Our application focus on DNA-dependent protein kinase (DNA-PK), a DNA repair factor with newly
identified role in RNA metabolism and a target of cancer therapy, and will use genetic, cell biology and single
molecule approaches to dissect the role of DNA-PK during lymphoma and leukemia-genesis and therapy.
Genomic instability is the hallmark of human cancer. The Non-Homologous End-Joining (NHEJ) is a major DNA
double-strand breaks (DSBs) repair pathway and is required for physiological gene-rearrangements and
oncogenic chromosomal translocations in developing lymphocytes. DNA-PK, composed of KU70-KU80
heterodimer (KU) and the large catalytic subunit (DNA-PKcs), is a NHEJ factor critical for both end-processing
(e.g., hairpin opening) and end-ligation during NHEJ. DNA-PKcs inhibitors is in phase I/IIa clinical trials for cancer
therapy. During NHEJ, KU binds to DNA ends, recruits and activates DNA-PKcs. Loss of DNA-PKcs abrogate
Artemis endonuclease mediated end-processing without abolishing end-ligation. We showed that expression of
kinase-dead (KD) (DNA-PKcsKD/KD) abrogates end-ligation without affecting end-processing, uncovering an end-
protection role of DNA-PKcs that is regulated by its own kinase activity. End-processing in DNA-PKcsKD/KD mice
is blocked by ATM inhibition, indicating end-processing requires DNA-PKcs protein, and the kinase activity from
either DNA-PKcs or the related ATM kinase in vivo. DNA-PKcs is the best characterized substrate of DNA-PK
and can also be phosphorylated by ATM. Mice carrying phosphorylation-deficient (DNA-PKcs5A/5A) DNA-PKcs
display mild end-ligation defects and are sensitive to ATM inhibition. Thus, we propose that once assembled on
KU-bound DNA, DNA-PK phosphorylation regulates end-processing and eventually the release of DNA-PKcs to
licence end-ligation. Moreover, we found that Ku can also direct the assembly of DNA-PKcs on structured RNA
(e.g., rRNA and snoRNA), where phosphorylation defective (DNA-PK5A) or KD DNA-PKcs (DNA-PKcsKD/KD)
blocks rRNA processing, protein translation, and erythropoiesis, leading to Trp53-dependent bone marrow failure.
These findings uncovered a NHEJ-independent role of DNA-PK in mammals. And two-third of DNA-
PKcsKD/KDTrp53-/- mice succumbed to ribosomal stress induced myeloid leukemia and one-third died of
lymphomas with IgH-Myc translocations, highlighting the critical role of DNA-PK in tumor suppression. Based on
these and other findings, we hypothesize that DNA-PKcs kinase activity and auto-phosphorylation regulates
KU-dependent assembly of DNA-PK on DNA and RNA to suppress lymphoma and leukemia genesis. To
test this, we will 1) characterize and compare KU and DNA-PK dynamics on RNA vs DNA; 2) elucidate how KU-
depletion impact RNA processing in human cells; 3) determine the physiological function of KU80 C-terminal
domain and tail in lymphoma and leukemia genesis and the recruitment and stabilization of DNA-PKcs. The
results will reveal the regul...

## Key facts

- **NIH application ID:** 10853032
- **Project number:** 5R01CA275184-03
- **Recipient organization:** COLUMBIA UNIVERSITY HEALTH SCIENCES
- **Principal Investigator:** Shan Zha
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2024
- **Award amount:** $471,356
- **Award type:** 5
- **Project period:** 2022-07-01 → 2027-06-30

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10853032

## Citation

> US National Institutes of Health, RePORTER application 10853032, The role of DNA-PKcs in DNA repair, lymphocyte development, RNA metabolism and tumor suppression (5R01CA275184-03). Retrieved via AI Analytics 2026-05-24 from https://api.ai-analytics.org/grant/nih/10853032. Licensed CC0.

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