Role of DNA damage sensing and TP53 function as modulators of sensitivity to calicheamicin-based antibody-drug conjugates (ADCs) for acute leukemia Free

Background: ADCs have long been pursued to improve outcomes in acute leukemia, with focus on CD33 (for acute myeloid leukemia [AML]) and CD22 (for B-acute lymphoblastic leukemia [B-ALL]). Better survival of some patients with the CD33 ADC, gemtuzumab ozogamicin (GO), and the CD22 ADC, inotuzumab ozogamicin (InO), validate these efforts, but these ADCs are ineffective in many others. Both GO and InO deliver a toxic calicheamicin (CLM) derivative, which induces DNA damage and, ultimately, cell death. As the resistance mechanisms to GO/InO remain incompletely understood, we performed a genome-wide clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 screen to identify CLM sensitivity genes.

Methods: Human acute leukemia cell lines (ML-1, OCI-AML3, and K562) were transduced with the Brunello CRISPR/Cas9 knockout library, exposed to CLM, and surviving cells were analyzed to identify differentially affected genes. For validation, the TP53 status was assessed in 13 human acute leukemia cell lines, and sublines with TP53 knockout (TP53^KO) derived in 5 TP53^WT cell backgrounds. CLM-induced cytotoxicity was assessed in vitro in the presence/absence of a Mouse Double Minute 2 homolog (MDM2) inhibitor (idasanutlin), Ataxia-Telangiectasia Mutated (ATM) inhibitor (AZD1390, lartesertib), Ataxia Telangiectasia and Rad3-related (ATR) inhibitor (ceralasertib), Checkpoint Kinase 1 and Checkpoint Kinase 1 (Chk1/Chk2) inhibitors (prexasertib, BML-277), and poly(ADP-ribose) polymerase (PARP) inhibitor (veliparib). After 72 hours of drug exposure, cell numbers and dead cells, using 4',6-diamidino-2-phenylindole (DAPI), were enumerated flow cytometrically.

Results: The drug screen identified several DNA damage pathway regulation genes in ML-1 cells, including TP53 (as highest ranked hit overall), PPP6R1, FBXW7, CHEK2, CDKN1A, DNTTIP1, and CSNK2A1 as conferring sensitivity to CLM. These findings were confirmed in OCI-AML3 and K562 cells (for TP53) and OCI-AML3 cells (for CHEK2). Across 13 acute leukemia cell lines, the six TP53-mutant cell lines (TP53^MUT) were 10- to 1000-fold less sensitive to CLM than the seven TP53^WT cell lines (at 1 ng/mL of CLM: 20±8% vs. 73±3% ΔDAPI+ cells). To test the relationship between TP53 function and CLM sensitivity further, we derived TP53^WT/KO syngeneic cell line pairs. When exposed to 0.05-0.2 ng/mL CLM, the five TP53^KO cells had an average decrease of 12±2% ΔDAPI+ cells when compared to their WT counterparts. Because TP53 and CHEK2 are in the same cellular response to DNA damage pathway as MDM2 and ATM, we next tested whether inhibitors of these pathways regulated response to CLM. Indeed, in TP53^WT cells, the MDM2 inhibitor and p53 activator, idasanutlin, enhanced CLM cytotoxicity, demonstrating that decoupling of cells from MDM2-p53 regulation could sensitize leukemia cells to CLM. For instance, in the seven TP53^WT cell lines, cytotoxicity increased from 28±9% ΔDAPI+ cells (CLM alone at 0.05 ng/mL) to 60±5% ΔDAPI+ cells when idasanutlin (0.04 µM) was added. In contrast, TP53^MUT cells were idasanutlin resistant, and even at very high doses, no combinatory effect was seen with CLM. Next, we found the ATM inhibitor, AZD1390, also enhanced CLM efficacy. However, this effect was independent of the TP53 status. For example, in TP53^WT cell lines, cytotoxicity increased from 17±5% with 0.05 ng/mL CLM alone to 48±8% ΔDAPI+ cells with the addition of 0.25-2.5 µM of AZD1390; across TP53^MUT cell lines, average cytotoxicity increased from 23±10% with 0.5 ng/mL of CLM to 47±4% ΔDAPI+ cells with the addition of 1-2.5 µM of AZD1390. Testing of a second ATM inhibitor, lartesertib, yielded qualitatively similar results. 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.

Conclusions: Our studies identify ATM, MDM2, and TP53 as key modulators of CLM-induced cytotoxicity in acute leukemia cells. These results support further evaluation of combination therapies with corresponding small molecule inhibitors (currently pursued in patients with other cancers) toward possible clinical testing as novel strategies to increase the efficacy of CLM-based ADCs such as GO and InO.