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IN VIVO PDX CRISPR/CAS9 SCREENS REVEAL MUTUAL THERAPEUTIC TARGETS TO OVERCOME HETEROGENEOUS ACQUIRED CHEMO-RESISTANCE
Author(s): ,
Anna-Katharina Wirth
Affiliations:
Apoptosis in Hematopoietic stem cells,Helmholtz Zentrum München, German Research Center for Environmental Health (HMGU),Munich,Allemagne;Apoptosis in Hematopoietic stem cells,Helmholtz Zentrum München, German Research Center for Environmental Health (HMGU),Munich,Deutschland;Apoptosis in Hematopoietic stem cells,Helmholtz Zentrum München, German Research Center for Environmental Health (HMGU),Muni
,
Lucas Wange
Affiliations:
Anthropology and Human Genomics, Faculty of Biology,Ludwig Maximilian University (LMU),Martinsried,Allemagne;Anthropology and Human Genomics, Faculty of Biology,Ludwig Maximilian University (LMU),Martinsried,Deutschland;Anthropology and Human Genomics, Faculty of Biology,Ludwig Maximilian University (LMU),Martinsried,Germania;Anthropology and Human Genomics, Faculty of Biology,Ludwig Maximilian Un
,
Sebastian Vosberg
Affiliations:
Clinical Division of Oncology, Department of Internal Medicine,Medical University of Graz,Graz,Autriche;Clinical Division of Oncology, Department of Internal Medicine,Medical University of Graz,Graz,Österreich;Clinical Division of Oncology, Department of Internal Medicine,Medical University of Graz,Graz,Austria;Clinical Division of Oncology, Department of Internal Medicine,Medical University of Gr
,
Ashok Kumar Jayavelu
Affiliations:
Department of Proteomics and Signal Transduction,Max Planck Institute of Biochemistry,Martinsried,Allemagne;Department of Proteomics and Signal Transduction,Max Planck Institute of Biochemistry,Martinsried,Deutschland;Department of Proteomics and Signal Transduction,Max Planck Institute of Biochemistry,Martinsried,Germania;Department of Proteomics and Signal Transduction,Max Planck Institute of Bi
,
Wolfgang Enard
Affiliations:
Anthropology and Human Genomics, Faculty of Biology,Ludwig Maximilian University (LMU),Martinsried,Allemagne;Anthropology and Human Genomics, Faculty of Biology,Ludwig Maximilian University (LMU),Martinsried,Deutschland;Anthropology and Human Genomics, Faculty of Biology,Ludwig Maximilian University (LMU),Martinsried,Germania;Anthropology and Human Genomics, Faculty of Biology,Ludwig Maximilian Un
,
Tobias Herold
Affiliations:
Department of Medicine III, and Laboratory for Leukemia Diagnostics,Ludwig Maximilian University (LMU),Munich,Allemagne;Department of Medicine III, and Laboratory for Leukemia Diagnostics,Ludwig Maximilian University (LMU),Munich,Deutschland;Department of Medicine III, and Laboratory for Leukemia Diagnostics,Ludwig Maximilian University (LMU),Munich,Germania;Department of Medicine III, and Laborat
Irmela Jeremias
Affiliations:
Apoptosis in Hematopoietic stem cells,Helmholtz Zentrum München, German Research Center for Environmental Health (HMGU),Munich,Allemagne;Apoptosis in Hematopoietic stem cells,Helmholtz Zentrum München, German Research Center for Environmental Health (HMGU),Munich,Deutschland;Apoptosis in Hematopoietic stem cells,Helmholtz Zentrum München, German Research Center for Environmental Health (HMGU),Muni
(Abstract release date: 05/12/22) EHA Library. Wirth A. 06/11/22; 356970; S105
Anna-Katharina Wirth
Anna-Katharina Wirth
Contributions
Abstract
Presentation during EHA2022: All Oral presentations will be presented between Friday, June 10 and Sunday, June 12 and will be accessible for on-demand viewing from Monday, June 20 until Monday, August 15, 2022 on the Congress platform.

Abstract: S105

Type: Oral Presentation

Session title: From biology to preclinical models in ALL

Background
Acquired resistance to conventional polychemotherapy regimens leads to relapse and poor prognosis, and remains a major unmet clinical need. 

Aims
To identify therapeutic options to overcome acquired chemo-resistance.

Methods

We studied acute lymphoblastic leukemia (ALL) as model disease and combined long-term in vivo treatment in orthotopic patient-derived xenograft (PDX) models with multi-omics profiling and functional genomic CRISPR/Cas9 screens. 

Results

We adapted conventional chemotherapeutic protocols to allow treatment of mice for up to 18 consecutive weeks, using a combination of the widely used drugs cyclophosphamide and vincristine. Three luciferase-transgenic PDX models were monitored by repetitive in vivo imaging. Polychemotherapy strongly reduced PDX ALL cells within the first weeks, proving initial sensitivity of the sample. Under continuous treatment, tumor load persisted in mice at the level of minimal residual disease for several weeks, until tumors resumed growth despite treatment, indicating acquired resistance. 

In an exemplary PDX model, eight resistant derivatives were generated in replicate mice and characterized individually. Genomic profiling revealed profound genomic heterogeneity between distinct derivatives; individual resistant derivatives acquired different copy number alterations in regions associated with resistance and distinct point mutations, e.g. in TP53. In contrast to genomic heterogeneity, transcriptome and proteome profiling identified a group of genes differentially expressed between sensitive and resistant cells, but similar across all derivatives. 

To gain insights into underlying mechanisms and to identify therapeutic targets to overcome acquired resistance, a customized CRISPR/Cas9 in vivo dropout screen was performed in individual resistant PDX derivatives, to test the relevance of around 200 candidate genes under treatment. Among others, sgRNAs targeting BCL2BRIP1 or COPS2 dropped out in the in vivo screen specifically during treatment; single knockout experiments confirmed that knockout of either BCL2, BRIP1 or COPS2 re-sensitized PDX ALL cells towards chemotherapy. Of direct translational relevance, treatment of mice with the BCL2 inhibitor ABT-199 sensitized resistant PDX cells towards polychemotherapy in vivo. Interestingly, BCL2 inhibition restored treatment response in resistant derivatives independently from the highly diverse underlying genetic alterations, e.g., in clones with and without mutation in TP53.

Conclusion
Taken together, we established a highly clinically relevant PDX in vivo model of acquired resistance to conventional chemotherapy. Using this model, we demonstrate that heterogeneous genomic alterations evolved in parallel in replicate mice, which could be overcome by a single therapeutic approach to re-sensitize tumors towards conventional chemotherapy.

Keyword(s): B cell acute lymphoblastic leukemia, Chemoresistance, Xenotransplantation

Presentation during EHA2022: All Oral presentations will be presented between Friday, June 10 and Sunday, June 12 and will be accessible for on-demand viewing from Monday, June 20 until Monday, August 15, 2022 on the Congress platform.

Abstract: S105

Type: Oral Presentation

Session title: From biology to preclinical models in ALL

Background
Acquired resistance to conventional polychemotherapy regimens leads to relapse and poor prognosis, and remains a major unmet clinical need. 

Aims
To identify therapeutic options to overcome acquired chemo-resistance.

Methods

We studied acute lymphoblastic leukemia (ALL) as model disease and combined long-term in vivo treatment in orthotopic patient-derived xenograft (PDX) models with multi-omics profiling and functional genomic CRISPR/Cas9 screens. 

Results

We adapted conventional chemotherapeutic protocols to allow treatment of mice for up to 18 consecutive weeks, using a combination of the widely used drugs cyclophosphamide and vincristine. Three luciferase-transgenic PDX models were monitored by repetitive in vivo imaging. Polychemotherapy strongly reduced PDX ALL cells within the first weeks, proving initial sensitivity of the sample. Under continuous treatment, tumor load persisted in mice at the level of minimal residual disease for several weeks, until tumors resumed growth despite treatment, indicating acquired resistance. 

In an exemplary PDX model, eight resistant derivatives were generated in replicate mice and characterized individually. Genomic profiling revealed profound genomic heterogeneity between distinct derivatives; individual resistant derivatives acquired different copy number alterations in regions associated with resistance and distinct point mutations, e.g. in TP53. In contrast to genomic heterogeneity, transcriptome and proteome profiling identified a group of genes differentially expressed between sensitive and resistant cells, but similar across all derivatives. 

To gain insights into underlying mechanisms and to identify therapeutic targets to overcome acquired resistance, a customized CRISPR/Cas9 in vivo dropout screen was performed in individual resistant PDX derivatives, to test the relevance of around 200 candidate genes under treatment. Among others, sgRNAs targeting BCL2BRIP1 or COPS2 dropped out in the in vivo screen specifically during treatment; single knockout experiments confirmed that knockout of either BCL2, BRIP1 or COPS2 re-sensitized PDX ALL cells towards chemotherapy. Of direct translational relevance, treatment of mice with the BCL2 inhibitor ABT-199 sensitized resistant PDX cells towards polychemotherapy in vivo. Interestingly, BCL2 inhibition restored treatment response in resistant derivatives independently from the highly diverse underlying genetic alterations, e.g., in clones with and without mutation in TP53.

Conclusion
Taken together, we established a highly clinically relevant PDX in vivo model of acquired resistance to conventional chemotherapy. Using this model, we demonstrate that heterogeneous genomic alterations evolved in parallel in replicate mice, which could be overcome by a single therapeutic approach to re-sensitize tumors towards conventional chemotherapy.

Keyword(s): B cell acute lymphoblastic leukemia, Chemoresistance, Xenotransplantation

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