GENOME-WIDE CRISPR-CAS9 SCREEN REVEALS SYNTHETIC LETHALITY OF WNT PATHWAY ACTIVATION AND ASPARAGINASE IN DRUG-RESISTANT ACUTE LEUKEMIAS
Author(s): ,
Laura Hinze
Affiliations:
Department of Pediatric Hematology and Oncolgy,Hannover Medical School,Hannover,Germany
,
Maren Pfirrmann
Affiliations:
Radboud University,Nijmegen,Netherlands
,
Salmaan Karim
Affiliations:
Division of Pediatric Hematology and Oncology,Boston Children's Hospital,Boston,United States
,
James Degar
Affiliations:
Division of Pediatric Hematology and Oncology,Boston Children's Hospital,Boston,United States
,
Connor McGuckin
Affiliations:
Division of Pediatric Hematology and Oncology,Boston Children’s Hospital,Boston,United States
,
Divya Vinjamur
Affiliations:
Division of Pediatric Hematology and Oncology,Boston Children’s Hospital,Boston,United States
,
Joshua Sacher
Affiliations:
Broad Institute of Massachusetts Institute of Technology and Harvard University,Cambridge,United States
,
Esteban Orellana
Affiliations:
Division of Pediatric Hematology and Oncology,Boston Children’s Hospital,Boston,United States
,
Richard Gregory
Affiliations:
Division of Pediatric Hematology and Oncology,Boston Children’s Hospital,Boston,United States
,
Daniel Bauer
Affiliations:
Division of Pediatric Hematology and Oncology,Boston Children’s Hospital,Boston,United States
,
Florence Wagner
Affiliations:
Broad Institute of Massachusetts Institute of Technology and Harvard University,Cambridge,United States
,
Kimberly Stegmaier
Affiliations:
Division of Pediatric Hematology and Oncology,Boston Children’s Hospital,Boston,United States
,
Martin Stanulla
Affiliations:
Department of Pediatric Hematology and Oncology,Hannover Medical School,Hannover,Germany
Alejandro Gutierrez
Affiliations:
Division of Pediatric Hematology and Oncology,Boston Children’s Hospital,Boston,United States
EHA Library. Hinze L. Jun 15, 2019; 267332; S859
Laura Hinze
Laura Hinze
Contributions
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Abstract

Abstract: S859

Type: Oral Presentation

Presentation during EHA24: On Saturday, June 15, 2019 from 12:15 - 12:30

Location: Hall E103

Background

Intensification of asparaginase-based therapy has improved outcomes for several subtypes of acute leukemia, but the development of treatment resistance has a poor prognosis, and effective therapeutic options are lacking for many of these patients.

Aims

We hypothesized, from the concept of synthetic lethality, that asparaginase-resistant leukemia cells harbor gain-of-fitness alterations that could be exploited therapeutically.

Methods

To identify molecular pathways that promote leukemic cell fitness upon treatment with asparaginase, we performed a genome-wide CRISPR-Cas9 loss-of-function genetic screen in the asparaginase-resistant T-ALL cell line CCRF-CEM. Cas9-expressing cells were transduced with the GeCKO genome-wide guide RNA library, treated with either vehicle or low-dose asparaginase, and guide RNA representation was assessed.

Results

Our internal positive control, asparagine synthetase, was the gene most significantly depleted in asparaginase-treated cells (RRA significance score = 1.56 x 10-7), followed closely by two regulators of Wnt signaling, NKD2 and LGR6 (RRA score = 6 x 10-6 and 2.19 x 10-5). Knockdown of NKD2 or LGR6 increased levels of active β-catenin, as well as the activity of a TopFLASH reporter of canonical Wnt/β-catenin transcriptional activity (P < 0.0001), indicating that both genes are negative regulators of Wnt signaling in these cells. Loss of NDK2 or LGR6 profoundly sensitized several acute leukemia cell lines to asparaginase (P < 0.0001), and potentiated asparaginase-induced apoptosis (P < 0.0001). Thus, Wnt pathway activation sensitizes leukemic cells to asparaginase.  

Wnt signaling inhibits glycogen synthase kinase 3 (GSK3) activity, prompting us to test whether pharmacologic GSK3 inhibition can enhance asparaginase induced cytotoxicity. Treatment with CHIR99021 (an ATP-competitive inhibitor of GSK3α and GSK3β) induced profound asparaginase sensitization in a panel of treatment-resistant acute leukemia cell lines, including T-ALL, MLL-rearranged AML and hypodiploid B-ALL (P < 0.0001), but not in normal hematopoietic progenitors.

Sensitization to asparaginase was independent of β-catenin and mTOR, but instead mediated by Wnt-dependent stabilization of proteins (Wnt/STOP), which inhibits GSK3-dependent protein ubiquitination and proteasomal degradation (Acebron et al. Mol Cell 2014). Proteasomal degradation is a catabolic source of amino acids, and we found that Wnt/STOP activation promoted leukemic cell death by exacerbating asparaginase-induced depletion of asparagine (P=0.017). Indeed, expression of a hyperactive PSMA4 proteasomal subunit that broadly increases proteasomal degradation (Choi et al. Nat Comm. 2016) completely blocked Wnt-induced sensitization to asparaginase (P < 0.0001).

To explore the therapeutic potential of our findings, we focused on GSK3 as a therapeutic target. Despite redundancy of GSK3α and GSK3β for many of their biologic functions, we found that depletion of GSK3α fully phenocopied Wnt-induced asparaginase sensitization (P < 0.0001), whereas GSK3β inhibition had no effect. Thus, we tested the combination of the GSK3α selective inhibitor BRD0705 (Wagner et al. Sci Transl Med 2018) and asparaginase in NRG mice, which were injected with patient-derived xenografts (PDXs) from different acute leukemia subtypes. All PDXs proved completely refractory to asparaginase monotherapy, but the combination was highly efficacious (Fig.1, < 0.0001).

Conclusion

These results provide a compelling rationale for clinical trials testing asparaginase in combination with GSK3α inhibitors for the treatment of acute leukemias.

Session topic: 1. Acute lymphoblastic leukemia - Biology & Translational Research

Keyword(s): Acute leukemia, Asparaginase, Drug resistance

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