Related Experiment Video
Updated: Jul 9, 2025
Performing an In Vitro Genome-Wide CRISPR Knockout Screen in Chimeric Antigen Receptor T Cells
Published on: January 31, 2025
Leukemia-intrinsic determinants of CAR-T response revealed by iterative in vivo genome-wide CRISPR screening
Azucena Ramos1,2, Catherine E Koch1,2, Yunpeng Liu-Lupo1,2
1Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, MA, USA.
Abstract:
CAR-T therapy is a promising, novel treatment modality for B-cell malignancies and yet many patients relapse through a variety of means, including loss of CAR-T cells and antigen escape. To investigate leukemia-intrinsic CAR-T resistance mechanisms, we performed genome-wide CRISPR-Cas9 loss-of-function screens in an immunocompetent murine model of B-cell acute lymphoblastic leukemia (B-ALL) utilizing a modular guide RNA library. We identified IFNγR/JAK/STAT signaling and components of antigen processing and presentation pathway as key mediators of resistance to CAR-T therapy in vivo; intriguingly, loss of this pathway yielded the opposite effect in vitro (sensitized leukemia to CAR-T cells). Transcriptional characterization of this model demonstrated upregulation of these pathways in tumors relapsed after CAR-T treatment, and functional studies showed a surprising role for natural killer (NK) cells in engaging this resistance program. Finally, examination of data from B-ALL patients treated with CAR-T revealed an association between poor outcomes and increased expression of JAK/STAT and MHC-I in leukemia cells. Overall, our data identify an unexpected mechanism of resistance to CAR-T therapy in which tumor cell interaction with the in vivo tumor microenvironment, including NK cells, induces expression of an adaptive, therapy-induced, T-cell resistance program in tumor cells.
Insights
CAR-T therapy resistance in B-cell acute lymphoblastic leukemia (B-ALL) involves tumor cells interacting with the microenvironment. This interaction triggers an adaptive resistance program, impacting treatment outcomes.
Area of Science:
- Immunology
- Oncology
- Genetics
Background:
- Chimeric antigen receptor T-cell (CAR-T) therapy shows promise for B-cell malignancies.
- Patient relapse occurs due to mechanisms like CAR-T cell loss and antigen escape.
Purpose of the Study:
- Investigate leukemia-intrinsic resistance mechanisms to CAR-T therapy.
- Identify genetic factors mediating resistance in vivo and in vitro.
Main Methods:
- Genome-wide CRISPR-Cas9 loss-of-function screens in a murine B-cell acute lymphoblastic leukemia (B-ALL) model.
- Transcriptional characterization of relapsed tumors.
- Functional studies involving natural killer (NK) cells.
- Analysis of patient data from CAR-T treated B-ALL patients.
Main Results:
- IFNγR/JAK/STAT signaling and antigen processing/presentation pathways mediate in vivo CAR-T resistance.
- Loss of these pathways sensitized leukemia to CAR-T cells in vitro.
- Relapsed tumors showed upregulation of these resistance pathways.
- NK cells play a role in activating this resistance program.
- Increased JAK/STAT and MHC-I expression in leukemia cells correlated with poor outcomes in patients.
Conclusions:
- Tumor cell interaction with the in vivo microenvironment, including NK cells, induces an adaptive, therapy-induced T-cell resistance program.
- This adaptive resistance mechanism contributes to CAR-T therapy failure in B-ALL.
