# DNA Damage Sensing and TP53 Function as Modulators of Sensitivity to Calicheamicin-Based Antibody–Drug Conjugates for Acute Leukemia

## TL;DR

This study explores how DNA damage pathways, especially TP53, influence the effectiveness of calicheamicin-based drugs in treating acute leukemia and identifies potential drug combinations to improve treatment outcomes.

## Contribution

The study identifies TP53, ATM, and MDM2 as key modulators of calicheamicin sensitivity and proposes combination therapies with small-molecule inhibitors to enhance treatment efficacy.

## Key findings

- TP53-mutant leukemia cells are significantly less sensitive to calicheamicin than TP53-wildtype cells.
- MDM2 and ATM inhibitors enhance calicheamicin-induced cytotoxicity in acute leukemia cells.
- Combination therapies with these inhibitors could improve the efficacy of calicheamicin-based ADCs.

## Abstract

Acute leukemias are difficult-to-treat blood cancers which many patients will eventually die from despite intensive chemotherapies and bone marrow transplantation. Antibodies that recognize proteins on leukemia cells and deliver a cell toxin (so-called antibody–drug conjugates) have been developed to improve these outcomes. Two such drugs, gemtuzumab ozogamicin (GO, for acute myeloid leukemia) and inotuzumab ozogamicin (IO, for acute lymphoblastic leukemia), have been approved for use in patients but are not always effective. In our laboratory research, we undertook genome-wide screening to discover genes that are associated with response or resistance to the toxin (calicheamicin) that both GO and IO contain. Our studies revealed the importance of several DNA damage pathway regulation genes for the anti-leukemia activity of calicheamicin, including TP53, ATM, and MDM2. Building on these data, we then identified several small-molecule inhibitors that increased GO/IO efficacy—findings that support further evaluation of these combination therapies with clinical testing.

Background/Objectives: Approved for treatment of acute leukemia, gemtuzumab ozogamicin (GO) and inotuzumab ozogamicin (InO) are antibody–drug conjugates (ADCs) that deliver a toxic calicheamicin (CLM) derivative. The resistance mechanisms to GO/InO remain incompletely understood. Methods: We performed a genome-wide clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 screen for CLM sensitivity genes, and then performed confirmatory cytotoxicity assays. Results: Several DNA damage pathway regulation genes were identified, most notably TP53. Across 13 acute leukemia cell lines, the six TP53-mutant cell lines (TP53MUT) were indeed 10- to 1000-fold less sensitive to CLM than the seven TP53WT cell lines. In five TP53WT/KO syngeneic cell line pairs we generated, TP53KO cells were significantly less sensitive to CLM than their TP53WT counterparts. In TP53WT but not TP53MUT cells, the MDM2 inhibitor and p53 activator, idasanutlin, enhanced CLM cytotoxicity, demonstrating that decoupling of cells from MDM2-p53 regulation sensitizes leukemia cells to CLM. The ATM inhibitors AZD1390 and lartesertib also significantly enhanced CLM efficacy but did so independent of the TP53 status. In contrast, neither an ATR inhibitor, Chk1/Chk2 inhibitor, Chk2 inhibitor, or a PARP inhibitor significantly impacted CLM-induced cytotoxicity across the thirteen cell lines. Together, our studies identify ATM, MDM2, and TP53—which are in the same cellular response to DNA damage pathway—as key modulators of CLM-induced cytotoxicity in acute leukemia cells. Conclusions: These results support further evaluation of combination therapies with corresponding small-molecule inhibitors (currently pursued for therapy of other cancers) toward clinical testing as novel strategies to increase the efficacy of CLM-based ADCs such as GO and InO.