Page 272 - Haematologica Vol. 107 - September 2022

LETTER TO THE EDITOR
Inducing synthetic lethality for selective targeting of acute myeloid leukemia cells harboring STAG2 mutations
Acute myeloid leukemia (AML) is a heterogeneous disorder and mostly incurable due to relapse and drug resistance. A key challenge is to target the fraction of more quiescent leukemic cells that are resistant to chemotherapeutic drugs.1 During recent years, several new small molecule in- hibitors have been developed to uniquely target disease- specific molecular events, such as the FLT3-ITD mutation. However, such targeted therapies have only met moderate clinical success due to drug resistance and relapse, which is associated with selection and expansion of malignant subclones that are not dependent on the initially targeted mutation.2 Thus, additional novel targeted therapeutic strategies are required for leukemia clearance. A particu- larly attractive approach is to harness specific genetic defi- ciencies of the tumor cells by targeting synthetic lethal interactions. In this case, a therapeutic effect can be reached even in tumors that are not critically dependent on the mutations that are targeted.
The cohesin protein complex forms a ring-like structure that holds sister chromatids together which is necessary for the proper segregation of chromosomes during mitosis. In addition, cohesin plays an essential role in DNA repair, genome organization and transcriptional regulation.3 The core structure comprises the three proteins RAD21, SMC3 and SMC1A. The fourth subunit consists of either one of two paralogous proteins: STAG1 or STAG2. Whole genome sequencing studies have identified a significant number of somatic mutations in cohesin genes with an accumulated mutation rate between 10% and 15% for AML and myelo- dysplastic syndrom (MDS).4 An even higher rate of cohesin mutations (around 50%) was observed in Down Syndrome associated childhood acute megakaryocytic leukemia (DS- AMKL).5 Cohesin mutations have loss-of-function con- sequences arguing for a tumor suppressor role of cohesin in the context of leukemia.4 Since cohesin is necessary for proper chromosomal segregation, mutations were first thought to promote tumor progression via genome insta- bility.6 However, the majority of the cohesin-mutated cancers are euploid suggesting that it is rather non-mitotic functions of cohesin such as transcriptional regulation and chromatin organization which are associated with leuke- mogenesis.7 With a mutation frequency of approximately 6% in AML and 18% in DS-AMKL, STAG2 is the predomi- nantly mutated gene among the cohesin genes.4 STAG2 mu- tations result in complete loss-of-function in males since these genes are located on the X chromosome.8 STAG2 knockdown promoted the in vitro expansion of umbilical cord blood (UCB)-derived hematopoietic stem and progen-
itor cells (HSPC) and enhanced the repopulating activity of human HSPC in xenograft recipients, demonstrating a di- rect functional association between STAG2 loss and dys- regulated hematopoiesis.9 Although STAG1- and STAG2-containing cohesin complexes might have distinct functions,10 they are redundant to ensure the chromatid cohesion during mitosis. Hence, STAG1-mediated mitotic dependency was observed in STAG2 knockout (KO) cell lines.11,12 Moreover, a recent study in knockout mice showed that the combined loss of STAG1 and STAG2, but not that of each gene alone, resulted in bone marrow aplasia and mor- tality, further supporting the existence of synthetic lethal interactions between STAG1 and STAG2.13 A majority of the cohesin mutations have a high variant allele frequency in- dicating that they occur relatively early in leukemogen- esis.4,14 Inducing synthetic lethality in AML harboring STAG2 mutations should thus eliminate most clones irrespective of any secondary mutations. Though, theoretically possible as a novel therapeutic application to STAG2 null AML, syn- thetic lethality from targeting STAG1 is yet to be demon- strated in primary human leukemic cells. Here, we provide a functional proof-of-concept demonstration for this ap- proach.
First, in order to demonstrate the existence of STAG1-me- diated mitotic dependency in primary human HSPC, we generated STAG2 null HSPC derived from UCB utilizing a CRISPR/Cas9 based knockin system. In brief, an early stop codon, followed by an open reading frame encoding en- hanced green fluorescent protein (eGFP) was inserted into the targeted STAG2 locus utilizing homology-directed repair (HDR) (Online Supplementary Figure S1A). With this system, successfully edited, eGFP expressing STAG2 null cells can be distinguished from unedited cells. As STAG2 is located in the X chromosome, we edited CD34+ cells from male do- nors to achieve successful STAG2 KO by monoallelic editing. Flow cytometry analysis revealed an eGFP frequency of 27% compared to mock control indicating efficient STAG2 editing and eGFP integration in HSPCs (Figure 1A and B). Further, Western blotting analysis of the eGFP expressing cells revealed a near complete loss of STAG2 protein, dem- onstrating that integration of the eGFP template success- fully knocked out STAG2 expression (Figure 1C). Sanger sequencing of the STAG2 locus in eGFP-positive cells re- veled a successful integration of the donor template at the expected locus (Online Supplementary Figure S1B). Alto- gether we successfully generated STAG2 KO human HSPC utilizing CRISPR-mediated HDR.
Next, in order to assess STAG1-mediated mitotic depend-
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