Abstract Medulloblastoma (MB) is the most common type of malignant pediatric brain cancer. Current standard of care (SOC) involves maximal safe resection and neuraxis radiotherapy and chemotherapy in individuals older than 3 years. To date, these cytotoxic SOC combined with craniospinal irradiation led to devastating neurocognitive and developmental deficits impacting quality of life for pediatric patients. The biological heterogeneity of MB is highlighted by the existence of four distinct molecular subgroups (WNT, SHH, Group 3, and Group 4). Group 3 and Group 4 have the poorest patient outcomes because of their aggressive, metastatic nature, and so often remain treatment refractory to SOC. Group 3 has a poor prognosis due to its high incidence of leptomeningeal spread and an overall survival rate of less than 50%. The cytotoxic nature and lack of response in specific subtypes to SOC underscores the urgent need for developing and translating novel treatment options including immunotherapies. In our earlier work, we have developed a therapy-adapted patient derived xenograft (PDX) model of the Group 3 MB as the tumor cells undergoes therapy in vitro and in vivo. N-glycocapture surfaceome profiling of the MB cells through this PDX model identified Integrin α5 (ITGA5) as one of the most differentially expressed targets found at recurrence when compared to engraftment and untreated timepoints. Through shRNA knockdown and small molecule inhibition, we identify ITGA5 expression marks a MB cell subpopulation with increased self-renewal ability both in vitro and in vivo. Access to recurrent MB (rMB) post-therapy allowed us to investigate the changes in the surfaceome of MB cells using proteomics profiling to identify promising rMB-specific targets for rational development of novel immunotherapies.
Supplementary Material 1: Fig. 1, related to Fig. 1 Glycocapture box blot (A) prior to and (B) after SUC2-normalization (yellow dot; NPVLAANSTQFRDPK peptide). (C) Hierarchical clustering heatmap based on Euclidean distance of differentially expressed proteins demonstrating close clustering of technical triplicates. (D) ITGA5 peptide tag intensity, (E)ITGA5 mRNA expression and (F) ITGB1 protein expression in Group 3 MB cells isolated at pre-determined timepoints through therapy. (G) Comparison of ITGA5 intensity to SUC2 demonstrating selective enrichment for ITGA5 in Group 3 MB through therapy. Bars represent mean of at least three technical replicates. *p ≤ 0.05, **p ≤ 0.001, ***p ≤ 0.0001, ****p ≤ 0.00001; unpaired t-test or one-way ANOVA with Sidak’s method for multiple comparisons. Fig. 2, related to Fig. 1. (A) A Venn diagram demonstrating the number of unique and overlapping surface proteins enriched at each stage of therapy when compared to their expression at engraftment and control timepoints. (B) A list of proteins used to generate the Venn diagram. R = post-radiation; RC = post-chemoradiotherapy; Re = relapse. Fig. 2, related to Fig. 2. In silico evaluation of ITGA5 in publicly available MB repository. (A) Kaplan-Meir curve demonstrating worse overall survival in patients (n = 288) with relative mRNA expression of ITGA5 over 4.6 (RMA- Normalized). (B) Transcriptional expression of ITGA5 of 632 patients across 12 MB subtypes described in Cavalli et al. and (C) based on age group affiliation. **p ≤ 0.001; one-way ANOVA with Dunnett’s method for multiple comparisons. Fig. 3, related to Fig. 3. Characterization and validation of ITGA5 KD. (A) mRNA expression of ITGA5 in HEK293FT cells post ITGA5 KD. (B) Microscopic images of HEK293FT cells post ITGA5 KD. Changes in (C)ITGA5 mRNA expression in reccurent Group 3 MB cells post ITGA5 KD. (D) Flow cytometric evaluation of changes in ITGA5 surface expression in HD-MB03-Re cells post ITGA5 KD. Bars represent mean of at least three technical replicates. *p ≤ 0.05, **p ≤ 0.001, ***p ≤ 0.0001, ****p ≤ 0.00001; unpaired t-test or one-way ANOVA with Sidak’s method for multiple comparisons. Fig. 4, related to Fig. 4. Selectivity characterization of dioscin in recurrent Group 3 MB cells and hNSCs. (A) Dose response curves and (B) corresponding IC50 concentrations and hill slopes of dioscin in hNSCs and three recurrent Group 3 MB lines. Points represent mean of three technical replicates, normalized to DMSO. Error bars represent standard error of the mean. IC50 and Hill slope values standardized to two decimal places. Fig. 5, related to Fig. 4. Expression of ITGA5 in healthy human tissue samples (A) ITGA5 protein levels as detected by whole cell proteomics in health tissues, reported by Wang et al. Protein intensity is reported as intensity-based absolute quantification (iBAQ) values, normalized using median centering across tissues. (B) ITGA5 protein expression as detected by antibody staining using the Human Proteome Atlas public repository. Staining strength corresponds to expression levels, including high (3), medium (2), low (1), and not detected (0). (C) mRNA expression of ITGA5 in various tissues according to Genotype-Tissue Expression (GTEx) dataset ( https://www.proteinatlas.org/ ); expressed in transcripts per million (TPM).
Abstract Medulloblastoma (MB) is the most common type of malignant pediatric brain cancer. The current standard of care (SOC) involves maximal safe resection and chemoradiotherapy in individuals older than 3 years, often leading to devastating neurocognitive and developmental deficits. Out of the four distinct molecular subgroups, Group 3 and 4 have the poorest patient outcomes due to the aggressive nature of the tumor and propensity to metastasize and recur post therapy. The toxicity of the SOC and lack of response in specific subtypes to the SOC underscores the urgent need for developing and translating novel treatment options including immunotherapies. To identify differentially enriched surface proteins that could be evaluated for potential future immunotherapeutic interventions, we leveraged N-glycocapture surfaceome profiling on Group 3 MB cells from primary tumor, through therapy, to recurrence using our established therapy-adapted patient derived xenograft model. Integrin 𝛼5 (ITGA5) was one of the most differentially enriched targets found at recurrence when compared to engraftment and untreated timepoints. In addition to being enriched at recurrence, shRNA-mediated knockdown and small molecule inhibition of ITGA5 have resulted in marked decrease in proliferation and self-renewal in vitro and demonstrated a survival advantage in vivo. Together, our data highlights the value of dynamic profiling of cells as they evolve through therapy and the identification of ITGA5 as a promising therapeutic target for recurrent Group 3 MB.
Abstract Glioblastoma (GBM) is the most common malignant primary brain tumor in adults. Despite an aggressive standard of care that includes maximally safe surgical resection, chemo-radiotherapy, median overall survival remains stagnant at 15 months. However, immunotherapeutic strategies have provided an exciting avenue of exploration to meet clinical need. Chimeric antigen receptor T-cell (CAR-T) therapy has shown promising results in liquid malignancies, but clinical trials in GBM targeting various tumor antigens have not shown durable clinical benefit. While this may be attributable to various tumor-intrinsic immune evasion strategies characteristic of GBM, little work has assessed whether the issue is due to the quality of the CAR-T treatment itself. Currently, CAR-Ts for clinical studies are manufactured in an autologous setting wherein T-cells are extracted from patients, engineered ex-vivo, and subsequently re-infused back. However, peripheral T-cells taken from untreated GBM patients have demonstrated qualitative and functional deficits, which may contribute to suboptimal treatment outcomes. Thus, we aimed to establish whether CAR-Ts generated from GBM patients would show reduced efficacy in comparison to healthy donors using our previously validated CD133 CAR-T. In this work, we show that in-spite of no inherent phenotypic differences, patient derived CAR-Ts shows pre-treatment exhaustion and upon preclinical evaluation using an orthotopic xenograft model of human GBM reduced survival advantage in autologous, patient-derived CD133-targeting CAR-T cell products was observed as compared to the controls. Transcriptomic analysis highlighted a decreased panel-wide enrichment in genes related to T cell and lymphocyte activation, lower prevalence of T cells (including Th1 and CD8+) and higher prevalence of exhausted CD8+ cells in T-cells products derived from GBM donors as compared to healthy donors. To overcome the functional and logistical considerations of autologous therapy, we additionally aimed to generate an “off-the-shelf” allogeneic CD133 CAR-T. Using CRISPR gene editing technology, we generated TCR-knockout CAR-T cells with comparable pre-clinical efficacy to our autologous models. In conclusion, this work highlights the need to reassess autologous CAR-T therapy for GBM and considers allogeneic approaches as promising alternatives. By addressing the inherent deficits in patient-derived CAR-Ts, allogeneic CD133 CAR-Ts may offer a more effective and logistically feasible therapeutic option for treating GBM. Citation Format: Muhammad Vaseem Shaikh, Sabra K. Salim, Jeffrey Wei, William T. Maich, Alisha A. Anand, Oliver Young Tang, Minomi K. Subapanditha, Yujin Suk, Manoj Singh, Zahra Alizada, Benjamin Brakel, Vassil Dimitrov, Zoya Tabunshchyk, Kevin Brown, Parvez Vora, Zev Binder, Chitra Venugopal, Jason Moffat, Sheila K. Singh. Generation of allogeneic CAR-T circumvents functional deficits in patient-derived autologous product for glioblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5241.
Abstract Glioblastoma (GBM) is the most common malignant brain tumor in adults, with a poor prognosis despite aggressive standard of care. Chimeric antigen receptor T-cell (CAR-T) therapy has shown promising results in liquid malignancies, but clinical trials in GBM targeting various tumor antigens have not shown durable clinical benefit. While this may be attributable to various tumor-intrinsic immune evasion strategies characteristic of GBM, little work has assessed whether the issue is due to the quality of the CAR-T treatment itself. Currently, CAR-Ts for clinical studies are manufactured in an autologous setting wherein T-cells are extracted from patients, engineered ex-vivo, and subsequently re-infused back. However, peripheral T-cells taken from untreated GBM patients have demonstrated qualitative and functional deficits, which may contribute to suboptimal treatment outcomes. Thus, we aimed to establish whether CAR-Ts generated from GBM patients would show reduced efficacy in comparison to healthy donors using our previously validated CD133 CAR-T. In this work, we show pre-treatment exhaustion and reduced survival advantage in autologous, patient-derived CD133-targeting CAR-T cell products using an orthotopic xenograft model of human GBM. To overcome the functional and logistical considerations of autologous therapy, we additionally aimed to generate an “off-the-shelf” allogeneic CD133 CAR-T. Using CRISPR gene editing technology, we generated TCR-knockout CAR-T cells with comparable pre-clinical efficacy to our autologous models. Ultimately, this work highlights the need to reassess autologous CAR-T therapy for GBM, and consider allogeneic approaches as biologically-informed therapeutic alternatives.
Medulloblastoma (MB) is the most common, malignant pediatric brain tumour comprised of four distinct molecular subgroups (WNT, SHH, Group 3, and Group 4). A subset of Group 3 MB tumors harbor focal amplifications of the MYC oncogene (MYC-G3MB) and are particularly prone to tumour recurrence and leptomeningeal spread due to their highly proliferative nature linked to its embryonic stem cell-of-origin and programing. Therefore, there is an urgent need for the development of therapeutic modalities that are safe and have potent antitumour efficacy against recurrent MB.
Methods
We discovered that LBR and TMPO, while present in all cells at the NE, is aberrantly presented to the cell surface in MYC-G3MB cell lines. Flow cytometry analysis identified highly abundant cell surface expression in all MYC-G3MB cell lines with low to no expression in human neural stem cells and other MB subgroups. Transcriptomic profiling of identifies significant NE protein enrichment in MB compared to normal tissue and with high expression during fetal development that dampens postnatally. Immunohistochemistry analysis of LBR on MB patient samples identified that positive LBR staining correlated with a significantly worse prognosis and was also identified to be enriched in recurrent tissue compared to their matched primary samples.
Results
Mechanistically, high resolution microscopy of endogenously tagged HALO-LBR and mNeon-TMPO reveals cell surface presentation to be linked to ER or ER-like vesicles that are trafficked to the cell surface. Transcriptomic analysis of LBR cell surface positive and negative cells identify that cell surface positivity is significantly linked to cell division and mitotic processes. LBR mislocalization is enriched following exposure to chemotherapy and radiation in vitro and in vivo and may act as a marker for the brain tumor initiating cells that seed recurrence. We then generated single domain antibodies (sdAb) to develop LBR and TMPO-specific CAR T cells. Validation using LBR KO MYC-G3MB cells show LBR sdAbs to have on-target binding and comparable affinity and avidity to commercial antibodies. Preliminary in vitro cytotoxicity assays of LBR-CAR T cell therapy reveal potent antitumour efficacy and CAR T cell activation following co-culture with of MYC-G3MB cells.
Conclusions
There is an undeniable paucity of recurrent tumor-specific targets for MB patients stemming from the lack of neoantigens and genomic aberrations in pediatric tumors. The preclinical development and validation of novel immunotherapeutic targets such as LBR for treatment of therapy-resistant, hyperproliferative MYC-G3MB provides a novel option for patients who otherwise face palliation (figure 1).
Histone deacetylase 6 (HDAC6) has been targeted in clinical studies for anticancer effects due to its role in oncogenic transformation and metastasis. Through a second-generation structure–activity relationship (SAR) study, the design, and biological evaluation of the selective HDAC6 inhibitor NN-390 is reported. With nanomolar HDAC6 potency, >200–550-fold selectivity for HDAC6 in analogous HDAC isoform functional assays, potent intracellular target engagement, and robust cellular efficacy in cancer cell lines, NN-390 is the first HDAC6-selective inhibitor to show therapeutic potential in metastatic Group 3 medulloblastoma (MB), an aggressive pediatric brain tumor often associated with leptomeningeal metastases and therapy resistance. MB stem cells contribute to these patients' poor clinical outcomes. NN-390 selectively targets this cell population with a 44.3-fold therapeutic margin between patient-derived Group 3 MB cells in comparison to healthy neural stem cells. NN-390 demonstrated a 45-fold increased potency over HDAC6-selective clinical candidate citarinostat. In summary, HDAC6-selective molecules demonstrated in vitro therapeutic potential against Group 3 MB.
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