Background Glioblastoma (GBM) is the most common primary malignant brain tumor in adults. Due to GBM displaying extreme heterogeneity and immune suppression, prognosis remains dismal. Glycoprotein nonmetastatic melanoma protein B (GPNMB) has previously been identified as a clinically relevant target in GBM while being absent in normal brain tissues and shown to be active in the tumor immune microenvironment.^1,2 Chimeric Antigen Receptor T-cells (CAR-Ts) fail clinical trials because multiple antigens will be required to eliminate all GBM subpopulations.^3,4 We previously proved CD133 to be an effective CAR-T target in GBM models.⁵

Methods Immunohistochemistry was performed on patient derived xenograft (PDX) brains and tissue microarrays of 16 patient matched primary/recurrent GBMs as well as 23 normal organ tissues. Whole cell proteomics was performed on 43 matched primary/recurrent GBM samples. Flow cytometry measured surface expression levels of CD133 and GPNMB to confirm CAR-T accessibility. CRISPR/Cas9 was used to eliminate expression in GBM lines to measure proliferation and mouse survival times. GPNMB knockout clones were generated in GL261 and engrafted in immunocompetent mice to examine single cell transcriptomes using sciRNAseq at endpoint. A second-generation CAR-T was developed to target GPNMB-expressing populations, and efficacy was interrogated using standard in vitro assays and PDX models.

Results GPNMB detected in residual tumors of PDX brains treated with CD133 CAR-Ts revealed it as a targetable subpopulation. Tissue microarrays and proteomics found GPNMB to be upregulated in recurrent GBMs compared to primary (p=0.0349 and p=0.0033 respectively), particularly in macrophage populations while being absent in normal tissues. Eliminating GPNMB in cell lines decreased proliferation (P≤0.001) and prolonged survival times in all mouse models (P≤0.01) indicating its functional relevance. GPNMB knockout clones displayed downregulation of hallmark signalling pathways of GBM such as PDGFR, TGF-beta, Integrins and Stats, as well as decreased innate/adaptive immune activation. CAR-T cytotoxicity and activation was observed in vitro and in vivo resulting in decreased tumor burden (P≤0.001) and increased survival times (P≤0.001). Ultimately a CD133+ population was observed in residual tumors of GPNMB treated mice at endpoint. Surface expression of CD133 and GPNMB revealed co-expression and distinct populations.

Conclusions We show GPNMB influences tumor intrinsic biology of GBM and is active in the tumor immune microenvironment. By targeting GPNMB along with CD133, combinatorial therapeutic regimens could target the cancer stem cell hierarchy and its supportive niche. We therefore plan to administer both CAR-T cells to provide better cytotoxic coverage and increase efficacy.

References

1. Kuan Chien-Tsun, Kenji Wakiya, Jeannette M Dowell, James E Herndon, David A Reardon, Michael W Graner, Gregory J Riggins, Carol J Wikstrand, and Darell D Bigner. Glycoprotein nonmetastatic melanoma protein B, a potential molecular therapeutic target in patients with glioblastoma multiforme. Clinical Cancer Research 2006;12:(7): 1970–82. https://doi.org/10.1158/1078-0432.ccr-05-2797

2. Feng X, Zhang L, Ke S, Liu T, Hao L, Zhao P, Tu W, & Cang S. High expression of GPNMB indicates an unfavorable prognosis in glioma: Combination of data from the GEO and CGGA databases and validation in tissue microarray. Oncology Letters, 2020;20(3), 2356–2368. https://doi.org/10.3892/ol.2020.11787

3. Brown Christine E, Brenda Aguilar, Renate Starr, Xin Yang, Wen-Chung Chang, Lihong Weng, Brenda Chang, et al. Optimization of IL13R?2-targeted chimeric antigen receptor T cells for improved anti-tumor efficacy against glioblastoma. Molecular Therapy 2018;26(1): 31–44. https://doi.org/10.1016/j.ymthe.2017.10.002

4. Goff Stephanie L, Richard A Morgan, James C Yang, Richard M Sherry, Paul F Robbins, Nicholas P Restifo, Steven A Feldman, et al. Pilot trial of adoptive transfer of chimeric antigen receptor–transduced T cells targeting EGFRVIII in patients with glioblastoma. Journal of Immunotherapy 2019;42(4) :126–35. https://doi.org/10.1097/cji.0000000000000260

5. Vora Parvez, Chitra Venugopal, Sabra Khalid Salim, Nazanin Tatari, David Bakhshinyan, Mohini Singh, Mathieu Seyfrid, et al. The rational development of CD133-targeting immunotherapies for glioblastoma. Cell Stem Cell 2020;26(6). https://doi.org/10.1016/j.stem.2020.04.008

Ethics Approval Human GBM samples were obtained from consenting patients, as approved by the Hamilton Health Sciences/McMaster Health Sciences Research Ethics Board(#07-366). All animal work was conducted by Animal Research Ethics Board (AREB)-approved Protocols (AUP#19-01-01).