Genome-wide CRISPR Screen Identifies DNTT as a Key Regulator of Inotuzumab Ozogamicin Response in B-cell Acute Lymphoblastic Leukemia

Inotuzumab Ozogamicin (InO) is an antibody-drug conjugate with striking efficacy in B-cell acute lymphoblastic leukemia (B-ALL). However, there is wide inter-patient variability in treatment response, and the genetic basis of this variation remains largely unknown. Using a genome-wide CRISPR screen, we discovered the loss of DNTT as a top driver of InO resistance. Our results directly show the effects of DNTT expression on H2AX phosphorylation, DNA damage response signaling, cell cycle arrest and mitochondrial apoptotic priming, ultimately conferring leukemia resistance to InO. Screening 196 primary human B-ALL samples for InO sensitivity confirmed the impact of DNTT expression on InO response. Additionally, low DNTT expression was found in the residual leukemia blasts post InO treatment in patients enrolled in the COG trial AALL1621. Collectively, we demonstrate that loss of DNTT diminishes B-ALL sensitivity to InO in vitro and promotes leukemia cell survival during InO treatment in vivo. Total RNA was purified from human B-ALL cell lines (Nalm6, REH and RS4:11 with WT or DNTT knockout genotype) with or without InO treatment by using the Qiagen Rneasy Mini Kit and subsquently used for RNA-sequencing.