A high-content screen of FDA approved drugs to enhance CAR T cell function: ingenol-3-angelate improves B7-H3-CAR T cell activity by upregulating B7-H3 on the target cell surface via PKCα activation
Abstract Background CAR T cell therapy is a promising approach to improve outcomes and decrease toxicities for patients with cancer. While extraordinary success has been achieved using CAR T cells to treat patients with CD19-positive malignancies, multiple obstacles have so far limited the benefit o...
| Main Authors: | , , , , , |
|---|---|
| Format: | Article |
| Language: | English |
| Published: |
BMC
2024-04-01
|
| Series: | Journal of Experimental & Clinical Cancer Research |
| Subjects: | |
| Online Access: | https://doi.org/10.1186/s13046-024-03022-x |
| _version_ | 1827291272827633664 |
|---|---|
| author | Ha Won Lee Carla O’Reilly Alex N. Beckett Duane G. Currier Taosheng Chen Christopher DeRenzo |
| author_facet | Ha Won Lee Carla O’Reilly Alex N. Beckett Duane G. Currier Taosheng Chen Christopher DeRenzo |
| author_sort | Ha Won Lee |
| collection | DOAJ |
| description | Abstract Background CAR T cell therapy is a promising approach to improve outcomes and decrease toxicities for patients with cancer. While extraordinary success has been achieved using CAR T cells to treat patients with CD19-positive malignancies, multiple obstacles have so far limited the benefit of CAR T cell therapy for patients with solid tumors. Novel manufacturing and engineering approaches show great promise to enhance CAR T cell function against solid tumors. However, similar to single agent chemotherapy approaches, CAR T cell monotherapy may be unable to achieve high cure rates for patients with difficult to treat solid tumors. Thus, combinatorial drug plus CAR T cell approaches are likely required to achieve widespread clinical success. Methods We developed a novel, confocal microscopy based, high-content screen to evaluate 1114 FDA approved drugs for the potential to increase expression of the solid tumor antigen B7-H3 on the surface of osteosarcoma cells. Western blot, RT-qPCR, siRNA knockdown and flow cytometry assays were used to validate screening results and identify mechanisms of drug-induced B7-H3 upregulation. Cytokine and cytotoxicity assays were used to determine if drug pre-treatment enhanced B7-H3-CAR T cell effector function. Results Fifty-five drugs were identified to increase B7-H3 expression on the surface of LM7 osteosarcoma cells using a novel high-content, high-throughput screen. One drug, ingenol-3-angelate (I3A), increased B7-H3 expression by up to 100%, and was evaluated in downstream experiments. Validation assays confirmed I3A increased B7-H3 expression in a biphasic dose response and cell dependent fashion. Mechanistic studies demonstrated that I3A increased B7-H3 (CD276) mRNA, total protein, and cell surface expression via protein kinase C alpha activation. Functionally, I3A induced B7-H3 expression enhanced B7-H3-CAR T cell function in cytokine production and cytotoxicity assays. Conclusions This study demonstrates a novel high-content and high-throughput screen can identify drugs to enhance CAR T cell activity. This and other high-content technologies will pave the way to develop clinical trials implementing rational drug plus CAR T cell combinatorial therapies. Importantly, the technique could also be repurposed for an array of basic and translational research applications where drugs are needed to modulate cell surface protein expression. |
| first_indexed | 2024-04-24T12:34:00Z |
| format | Article |
| id | doaj.art-67caf5f0c48247379501ee94399f1f1a |
| institution | Directory Open Access Journal |
| issn | 1756-9966 |
| language | English |
| last_indexed | 2024-04-24T12:34:00Z |
| publishDate | 2024-04-01 |
| publisher | BMC |
| record_format | Article |
| series | Journal of Experimental & Clinical Cancer Research |
| spelling | doaj.art-67caf5f0c48247379501ee94399f1f1a2024-04-07T11:34:24ZengBMCJournal of Experimental & Clinical Cancer Research1756-99662024-04-0143111310.1186/s13046-024-03022-xA high-content screen of FDA approved drugs to enhance CAR T cell function: ingenol-3-angelate improves B7-H3-CAR T cell activity by upregulating B7-H3 on the target cell surface via PKCα activationHa Won Lee0Carla O’Reilly1Alex N. Beckett2Duane G. Currier3Taosheng Chen4Christopher DeRenzo5Department of Chemical Biology and Therapeutics, St. Jude Children’s Research HospitalDepartment of Bone Marrow Transplantation and Cellular Therapy, St. Jude Children’s Research HospitalDepartment of Bone Marrow Transplantation and Cellular Therapy, St. Jude Children’s Research HospitalDepartment of Chemical Biology and Therapeutics, St. Jude Children’s Research HospitalDepartment of Chemical Biology and Therapeutics, St. Jude Children’s Research HospitalDepartment of Bone Marrow Transplantation and Cellular Therapy, St. Jude Children’s Research HospitalAbstract Background CAR T cell therapy is a promising approach to improve outcomes and decrease toxicities for patients with cancer. While extraordinary success has been achieved using CAR T cells to treat patients with CD19-positive malignancies, multiple obstacles have so far limited the benefit of CAR T cell therapy for patients with solid tumors. Novel manufacturing and engineering approaches show great promise to enhance CAR T cell function against solid tumors. However, similar to single agent chemotherapy approaches, CAR T cell monotherapy may be unable to achieve high cure rates for patients with difficult to treat solid tumors. Thus, combinatorial drug plus CAR T cell approaches are likely required to achieve widespread clinical success. Methods We developed a novel, confocal microscopy based, high-content screen to evaluate 1114 FDA approved drugs for the potential to increase expression of the solid tumor antigen B7-H3 on the surface of osteosarcoma cells. Western blot, RT-qPCR, siRNA knockdown and flow cytometry assays were used to validate screening results and identify mechanisms of drug-induced B7-H3 upregulation. Cytokine and cytotoxicity assays were used to determine if drug pre-treatment enhanced B7-H3-CAR T cell effector function. Results Fifty-five drugs were identified to increase B7-H3 expression on the surface of LM7 osteosarcoma cells using a novel high-content, high-throughput screen. One drug, ingenol-3-angelate (I3A), increased B7-H3 expression by up to 100%, and was evaluated in downstream experiments. Validation assays confirmed I3A increased B7-H3 expression in a biphasic dose response and cell dependent fashion. Mechanistic studies demonstrated that I3A increased B7-H3 (CD276) mRNA, total protein, and cell surface expression via protein kinase C alpha activation. Functionally, I3A induced B7-H3 expression enhanced B7-H3-CAR T cell function in cytokine production and cytotoxicity assays. Conclusions This study demonstrates a novel high-content and high-throughput screen can identify drugs to enhance CAR T cell activity. This and other high-content technologies will pave the way to develop clinical trials implementing rational drug plus CAR T cell combinatorial therapies. Importantly, the technique could also be repurposed for an array of basic and translational research applications where drugs are needed to modulate cell surface protein expression.https://doi.org/10.1186/s13046-024-03022-xIngenol-3-angelatePKCB7-H3CART cellOsteosarcoma |
| spellingShingle | Ha Won Lee Carla O’Reilly Alex N. Beckett Duane G. Currier Taosheng Chen Christopher DeRenzo A high-content screen of FDA approved drugs to enhance CAR T cell function: ingenol-3-angelate improves B7-H3-CAR T cell activity by upregulating B7-H3 on the target cell surface via PKCα activation Journal of Experimental & Clinical Cancer Research Ingenol-3-angelate PKC B7-H3 CAR T cell Osteosarcoma |
| title | A high-content screen of FDA approved drugs to enhance CAR T cell function: ingenol-3-angelate improves B7-H3-CAR T cell activity by upregulating B7-H3 on the target cell surface via PKCα activation |
| title_full | A high-content screen of FDA approved drugs to enhance CAR T cell function: ingenol-3-angelate improves B7-H3-CAR T cell activity by upregulating B7-H3 on the target cell surface via PKCα activation |
| title_fullStr | A high-content screen of FDA approved drugs to enhance CAR T cell function: ingenol-3-angelate improves B7-H3-CAR T cell activity by upregulating B7-H3 on the target cell surface via PKCα activation |
| title_full_unstemmed | A high-content screen of FDA approved drugs to enhance CAR T cell function: ingenol-3-angelate improves B7-H3-CAR T cell activity by upregulating B7-H3 on the target cell surface via PKCα activation |
| title_short | A high-content screen of FDA approved drugs to enhance CAR T cell function: ingenol-3-angelate improves B7-H3-CAR T cell activity by upregulating B7-H3 on the target cell surface via PKCα activation |
| title_sort | high content screen of fda approved drugs to enhance car t cell function ingenol 3 angelate improves b7 h3 car t cell activity by upregulating b7 h3 on the target cell surface via pkcα activation |
| topic | Ingenol-3-angelate PKC B7-H3 CAR T cell Osteosarcoma |
| url | https://doi.org/10.1186/s13046-024-03022-x |
| work_keys_str_mv | AT hawonlee ahighcontentscreenoffdaapproveddrugstoenhancecartcellfunctioningenol3angelateimprovesb7h3cartcellactivitybyupregulatingb7h3onthetargetcellsurfaceviapkcaactivation AT carlaoreilly ahighcontentscreenoffdaapproveddrugstoenhancecartcellfunctioningenol3angelateimprovesb7h3cartcellactivitybyupregulatingb7h3onthetargetcellsurfaceviapkcaactivation AT alexnbeckett ahighcontentscreenoffdaapproveddrugstoenhancecartcellfunctioningenol3angelateimprovesb7h3cartcellactivitybyupregulatingb7h3onthetargetcellsurfaceviapkcaactivation AT duanegcurrier ahighcontentscreenoffdaapproveddrugstoenhancecartcellfunctioningenol3angelateimprovesb7h3cartcellactivitybyupregulatingb7h3onthetargetcellsurfaceviapkcaactivation AT taoshengchen ahighcontentscreenoffdaapproveddrugstoenhancecartcellfunctioningenol3angelateimprovesb7h3cartcellactivitybyupregulatingb7h3onthetargetcellsurfaceviapkcaactivation AT christopherderenzo ahighcontentscreenoffdaapproveddrugstoenhancecartcellfunctioningenol3angelateimprovesb7h3cartcellactivitybyupregulatingb7h3onthetargetcellsurfaceviapkcaactivation AT hawonlee highcontentscreenoffdaapproveddrugstoenhancecartcellfunctioningenol3angelateimprovesb7h3cartcellactivitybyupregulatingb7h3onthetargetcellsurfaceviapkcaactivation AT carlaoreilly highcontentscreenoffdaapproveddrugstoenhancecartcellfunctioningenol3angelateimprovesb7h3cartcellactivitybyupregulatingb7h3onthetargetcellsurfaceviapkcaactivation AT alexnbeckett highcontentscreenoffdaapproveddrugstoenhancecartcellfunctioningenol3angelateimprovesb7h3cartcellactivitybyupregulatingb7h3onthetargetcellsurfaceviapkcaactivation AT duanegcurrier highcontentscreenoffdaapproveddrugstoenhancecartcellfunctioningenol3angelateimprovesb7h3cartcellactivitybyupregulatingb7h3onthetargetcellsurfaceviapkcaactivation AT taoshengchen highcontentscreenoffdaapproveddrugstoenhancecartcellfunctioningenol3angelateimprovesb7h3cartcellactivitybyupregulatingb7h3onthetargetcellsurfaceviapkcaactivation AT christopherderenzo highcontentscreenoffdaapproveddrugstoenhancecartcellfunctioningenol3angelateimprovesb7h3cartcellactivitybyupregulatingb7h3onthetargetcellsurfaceviapkcaactivation |