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1445 Genome-Wide CRISPR/Cas9 Screens Identify DDX19A/DDX19B As a Critical Regulator of Intrinsic Apoptosis By Regulating MCL1 mRNA Cellular Localization

Program: Oral and Poster Abstracts
Session: 605. Molecular Pharmacology and Drug Resistance: Lymphoid Neoplasms: Poster I
Hematology Disease Topics & Pathways:
Lymphoid Leukemias, ALL, Diseases, Lymphoid Malignancies
Saturday, December 9, 2023, 5:30 PM-7:30 PM

Tatsuya Terasaki, MD1*, Yuichiro Semba, MD, PhD2*, Kensuke Sasaki, MD, PhD2*, Kiyoko Miyata, PhD2*, Takuji Yamauchi, MD, PhD2*, Hiroshi Imanaga, MD2*, Fumihiko Nakao, MD3*, Shigeki Hirabayashi, MD, PhD2*, Jumpei Nogami, PhD2*, Koshi Akahane, MD, PhD4*, Takeshi Inukai, MD, PhD4*, Koichi Akashi, MD, PhD2* and Takahiro Maeda, MD, PhD2

1Kyushu University Graduate School of Medical Sciences, Fukuoka, FKO, Japan
2Kyushu University Graduate School of Medical Sciences, Fukuoka, Japan
3National Hospital Organization Kyushu Medical Center, Fukuoka, Japan
4Department of Pediatrics, School of Medicine, University of Yamanashi, Chuo, Japan

Despite recent advancements, the prognosis of relapsed/refractory acute lymphoblastic leukemia (ALL) remains dismal, necessitating novel therapies. Selinexor, an XPO1 (Exportin-1) inhibitor, shows effectiveness in multiple myeloma (MM), diffuse large B-cell lymphoma (DLBCL), and ALL mouse models. However, its broad target spectrum could lead to off-target toxicities, limiting its clinical utility.

To elucidate Selinexor's molecular mechanisms in ALL we used CRISPR-Cas9 screens in the presence of Selinexor on ALL cell lines NALM-6 and KOPN-49. Among 18 genes displaying synthetic lethality in both cell lines with Selinexor, we focused on paralogous genes DDX19A and DDX19B, critical in nucleocytoplasmic transport of mRNA and translation initiation by dissociating protein complexes bound to mRNA.

Competition assays showed prominent growth inhibitions when DDX19B was knocked out prior to DDX19A depletion in both NALM-6 and KOPN-49, while single KO of either DDX19A or DDX19B did not remarkably inhibit cell growth. Additionally, in MOLT-3 with low DDX19B expression, significant growth inhibitions were observed upon single KO of DDX19A and these phenotypes were canceled by the overexpression of DDX19B, suggesting functional redundancy between DDX19A and DDX19B.

Further, we generated MOLT-3 that stably express HA-FKBP12F36V-tagged DDX19A and knocked out endogenous DDX19A. Western blot analysis showed depletion of DDX19A by dTAGV-1 led to upregulate cleaved caspase-3 expression level within 4 hours, accompanied by a significant decrease in MCL1 protein levels in a BAX/BAK dependent manner. These phenotypes were rescued by the overexpression of MCL1, suggesting that the loss of DDX19A/19B induced intrinsic apoptosis through the downregulation of MCL1 expression.

Given that the depletion of DDX19A/19B led to the mislocalization of polyadenylated mRNA and that neither the stability of the MCL1 protein nor the translation of MCL1 mRNA was affected by this depletion, as confirmed by RNA-FISH, and Actinomycin-D and Cycloheximide chase assays respectively, we concluded that DDX19A/19B are crucial for the nucleocytoplasmic transport of MCL1 mRNA.

Furthermore, in exploring the relationship between the loss of DDX19A/19B and sensitivity to Selinexor, we found that combined treatment with Selinexor and the depletion of either DDX19A or DDX19B leads to a reduction in MCL1 protein, which can be rescued by overexpressing MCL1.

Through CRISPR-Cas9 screens, we identified DDX19A/19B, which are in a paralogous relationship, as potential therapeutic targets for ALL. Mechanistically, the loss of DDX19A/19B leads to impaired cytoplasmic mRNA transport, leading to the inhibition of novel synthesis and reduced expression of MCL1 protein, thereby inducing intrinsic apoptosis. Furthermore, we found that DDX19A/19B depletion exerts synergistic effects with Selinexor through the downregulation of MCL1 expression, suggesting that DDX19A/19B levels could serve as a biomarker for Selinexor treatment in ALL.

Disclosures: Akashi: Pfizer Japan Inc.: Honoraria; ONO PHARMACEUTICAL CO., LTD: Honoraria; Eisai Co., Ltd.: Honoraria; Takeda Pharmaceuticals Co,. Ltd.: Research Funding; CHUGAI PHARMACEUTICAL CO., LTD.: Honoraria, Research Funding; AbbVie Inc.: Honoraria, Research Funding; ASAHIKASEI PHARMA CORPORATION CO., LTD: Research Funding; Kyowa Kirin Co., Ltd.: Honoraria, Research Funding; Otsuka Pharmaceutical Co.,Ltd.: Research Funding; Bristol-Myers Squibb K.K.: Honoraria; Janssen Pharmaceutical K.K.: Honoraria.

*signifies non-member of ASH