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949 CRISPR/Cas9 Library Screens Identified Atp2a2 As an In Vivo Specific Tumor Suppressor in Myeloid Neoplasms

Program: Oral and Poster Abstracts
Type: Oral
Session: 602. Myeloid Oncogenesis: Basic: CRISPR Screens and Other New Approaches to Understanding Myeloid Neoplasia
Hematology Disease Topics & Pathways:
Fundamental Science, Research
Monday, December 9, 2024: 4:30 PM

Ruka Shimura, MSc1*, Moe Tamura, PhD2*, Keita Yamamoto, MD, PhD2*, Kohei Iida, PhD2*, Shuhei Asada, MD, PhD3, Emi Sugimoto, PhD2*, E. Christine Pietsch, PhD4, Barbara A. Weir4*, Ramona Crescenzo, PhD5*, Glenn S. Cowley4*, Ricardo M. Attar, PhD6*, Ulrike Philippar, PhD7, Satoshi Yamasaki, PhD8*, Taisei Hirouchi, PhD9*, Kaoru Uchimaru, MD, PhD9*, Toshio Kitamura, MD, PhD10,11 and Susumu Goyama, MD, PhD2*

1Graduate School of Frontier Sciences, The University of Tokyo, Minato-Ku, Japan
2Graduate School of Frontier Sciences, The University of Tokyo, Tokyo, Japan
3The Institute of Medical Science, The University of Tokyo, Tokyo, Japan
4Janssen Research and Development, Spring House, PA
5Janssen Research and Development, Beerse, Belgium
6Janssen Research & Development, Spring House, PA
7Janssen Research & Development, Beerse, Belgium
8Institute of Medical Science, University of Tokyo, Tokyo, Japan
9Laboratory of Tumor Cell Biology, Department of Computational Biology and Medical Sciences, Graduate School of Frontier Sciences, The University of Tokyo, Tokyo, Japan
10Graduate School of Pharmaceutical Sciences, The University of Tokyo, Tokyo, Japan
11Institute of Biomedical Research and Innovation, Foundation for Biomedical Research and Innovation at Kobe, Kobe, Japan

CRISPR/Cas9 library screens have revealed numerous oncogenes and tumor suppressors in malignant tumors. However, most of the genetic screens have been performed using in vitro culture assays, which may miss critical genes that specifically regulate tumorigenesis in vivo. To identify the therapeutic targets that specifically regulate tumorigenesis in vivo, we performed in vivo CRISPR/Cas9 library screens using two mouse models of myeloid tumors driven by MLL-AF9 and ASXL1/SETBP1 mutations (MA9 and cSAM cells, respectively). We ranked all genes according to in vivo specific essentiality, and identified a sarco/endoplasmic reticulum (ER) calcium-ATPase Atp2a2 (also known as SERCA2) as an in vivo specific tumor suppressor.

To validate the results of our screens, we examined the effect of individual depletion of Atp2a2 in MA9 and cSAM cells. Interestingly, Atp2a2 depletion in these myeloid tumor cells showed contrasting results between in vitro and in vivo. Consistent with the screening results, Atp2a2 depletion accelerated the development of myeloid tumors in vivo. In sharp contrast, Atp2a2 depletion inhibited the growth of MA9 and cSAM cells in vitro. We then examined the effect of Atp2a2 depletion in MA9 cells on responses to cytotoxic drugs, cytarabine and a p53 activating agent, DS-5272. Treatment of C57BL/6 mice bearing MA9 cells with cytarabine or DS-5272 induced an enrichment of Atp2a2-depleted cells in the bone marrow, whereas no enrichment of Atp2a2-depleted cells was observed in vitro, again indicating that Atp2a2 depletion confers resistance to cytotoxic therapies in MA9 cells specifically in vivo. These results suggest that Atp2a2 is an in vivo specific tumor suppressor in myeloid tumors.

Since ATP2A2 is known to play a key role in pumping Ca2+ from the cytosol into the ER lumen, we next investigated the effect of Atp2a2 depletion on Ca2+ homeostasis in MA9 cells. Atp2a2 depletion induced a decrease in ER Ca2+, an increase in cytosolic Ca2+, and an aberrant activation of Store-Operated Calcium Entry (SOCE) in response to the reduction in ER Ca2+ stores. Consequently, Atp2a2-depleted MA9 cells became dependent on Stim1 and Stim2, which play key roles in the SOCE pathway, indicating the essential role of SOCE in maintaining Ca2+ homeostasis in Atp2a2-depleted cells. The reduced ER Ca2+ also resulted in increased ER stress and downregulation of major histocompatibility complex classⅠ(MHC-I) in Atp2a2-depleted MA9 cells, which may promote immune evasion of myeloid tumors. Indeed, the proliferative advantage of Atp2a2-depleted MA9 cells in vivo was attenuated in the immunodeficient NSG mice and by the antibody-based CD8+ T-cells depletion in C57BL/6 mice. Regarding the differential influence of Atp2a2 depletion on AML growth in vitro and in vivo, we found that Atp2a2 depletion strongly induced cell cycle arrest in MA9 cells in the cytokine-rich in vitro culture, whereas it had little effect on the cell cycle in vivo. Thus, the context-dependent role of ATP2A2 in leukemogenesis could be partially explained by the reduced immunogenicity of Atp2a2-depleted cells and its differential impact on cell cycle progression in vitro and in vivo. Metabolomic analysis revealed that Atp2a2 depletion induced pro-tumorigenic metabolic alterations, such as enhanced purine metabolism and activation of the pentose phosphate pathway, which may also contribute to their increased leukemogenicity in vivo.

Finally, we investigated the association between reduced ER Ca2+ levels and prognosis in human AML using RNA-seq data from Beat AML [Nature Medicine 26, 1852-1858 (2020)]. Consistent with the results from mouse AML models, human AMLs with low SERCA (ATP2A1+ATP2A2+ATP2A3) and ER Ca2+ uptake (ATP2A1/2/3-ITPR1/2/3-RyR1/2/3) scores showed significantly worse prognosis than those with high SERCA and ER Ca2+ uptake scores (p = 0.0296 and 0.0009, respectively). Thus, human AMLs with low ER Ca2+ appear to be resistant to current standard treatments with cytotoxic drugs.

In conclusion, we performed the in vivo CRISPR/Cas9 library screens and identified Atp2a2 as a novel in vivo specific tumor suppressor in myeloid tumors. Deletion of Atp2a2 alters Ca2+ signaling, increases ER stress and confers resistance to cytotoxic drugs in myeloid tumors. Our data also suggest that SOCE is a synthetic lethal target for myeloid tumors with low ATP2A2 activity.

Disclosures: Pietsch: Janssen Research & Development: Current Employment. Weir: Janssen Research & Development: Current Employment. Crescenzo: Janssen Research & Development: Current Employment. Cowley: Janssen Research & Development: Current Employment. Attar: Johnson & Johnson: Current Employment, Current holder of stock options in a privately-held company. Philippar: Johnson&Johnson Innovative Medicine: Current Employment, Current holder of stock options in a privately-held company, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties. Uchimaru: Minophagen Pharmaceutical: Honoraria; Meiji-Seika Pharma: Honoraria; JIMRO: Honoraria; LSI Medience: Patents & Royalties; Daiichi-Sankyo: Honoraria, Research Funding.

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