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Key takeaways
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Children with group 3 medulloblastoma suffer significant morbidity and mortality
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Researchers found the drug combo CI-994 and anti-CD47 shows promise
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They unblock “don’t eat me pathways” that prevent macrophages in the immune system from consuming a tumor
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Future clinical trials will test its therapeutic efficacy against high-risk MB
Background: Children with group 3 medulloblastoma face poor outcomes
Medulloblastoma (MB) is the most common malignant pediatric brain tumor. These tumors are made up of four different molecular subgroups: Wingless (Wnt), Sonic hedgehog (Shh), group 3 and group 4.
The prognosis is poor for children with group 3 MB due to the lack of combination therapies to target the amplification of proto-oncogene MYC in these tumors, which is involved in cellular growth and proliferation. There’s a greater risk for metastatic dissemination (the tumor cell spreading into the leptomeningeal structures of the brain and spine), with 39% of children experiencing tumor metastasis and less than 45% achieving a 5-year overall survival rate. Most of these tumors recur metastatically, continuing or gaining MYC amplification at recurrence, and there’s a lack of second-line treatment options.
In this multi-center, international study, researchers investigated epigenetic inhibitors to identify potential treatments for children with therapy-resistant malignant tumors. Researchers from the Center for Cancer and Blood Disorders and the Neuro-Oncology Program at Children’s Hospital Colorado, including Siddhartha Mitra, PhD, Rajeev Vibhakar, MD, PhD, and Sujatha Venkatraman, PhD, worked in collaboration with the German Cancer Consortium (DKTK) University Hospital Dusseldorf.
Epigenic changes have been at the center of previous large studies because, unlike genes, they can be reversed and do not alter DNA. Past studies found that mutations and structural changes of chromatin modifiers, abnormal DNA methylation and histone modification signatures cause abnormal cancer epigenomes. These findings sparked growing interest in targeting epigenetic modifiers for cancer treatment, and several epigenetic modulators have undergone rigorous clinical trials.
Methods: Drug screenings seek to identify active medulloblastoma tumor inhibitors
In this study, investigators performed a primary screen to compare antitumoral activity of 78 epigenetic inhibitors in:
- 11 atypical teratoid/rhabdoid tumors (AT/RT)
- 14 MB
- 14 glioblastoma
To remove bias when evaluating selective activity emerging from biological differences, a secondary screen of 20 commercially available preclinical and clinically approved histone deacetylase inhibitors (HDACi) was performed using an in-house semi-automated platform.
In the secondary screening, the `study authors compared the median response (IC50 value) in one entity against the median of the other tested entities together to identify the inhibitors selectively active in one entity or subgroup.
Of the 78 inhibitors screened:
- 48 had median IC50 greater than 25 µM (negligible activity)
- 17 had significantly lower IC50 values in MB compared with glioblastoma and AT/RT in vitro
In those 17, 11 (65%) of the selective compounds were HDACi, demonstrating over-representation. After subdividing MB models by MYC status, the 11 inhibitors had significantly decreased IC50 values compared to MYC-driven MB with non-MYC-driven MB.
Study authors then analyzed epigenetic inhibitors exhibiting preferential sensitivity in MYC-driven MB, identifying:
- 32 with higher median activity in MYC-driven MB
- 13 significantly more active compared to other tested entities
- 40 with no preferential activity (group 2)
- 4 less active in MYC-driven MB when compared with the median response in the other tested entities (group 3)
Of these 9 (69%):
- 6 demonstrated selective activity when in glioblastoma or AT/RT models compared against other cell lines together
- 3 histone methyltransferase G9a inhibitors were preferentially active in glioblastoma cell lines (already demonstrating promise in glioblastoma therapy)
- 2 DNMTs inhibitors (5-azacytidine and fisetin) and Jumonji inhibitor GSKJ4 identified as selectively active in AT/RT models
Findings from the cross-entity screen highlighted the remarkable in vitro antitumoral potential of HDACi for MYC-driven MB.
Study authors performed a second screen, exclusively evaluating HDACi, using a cross-entity panel of brain tumor cell lines, including:
- 15 previously evaluated HDACi
- 5 FDA-approved drugs:
- Panobinostat
- Romidepsin
- Belinostat
- Entinostat
- Tubastatin A
For the FDA-approved drugs, vorinostat, panobinostat, romidepsin and belinostat showed substantial inhibition of cell viability across all three tumor entities. Panobinostat and romidepsin had low nanomolar IC50 values.
For the previously evaluated HDACi, valproic acid, phenylbutyrate and MC 1568 did not inhibit cell viability at maximal concentration of 25 µM in any screened cell lines. None of the previously evaluated HDACi were electively active against glioblastoma or AT/RT cell lines in vitro.
Of the 20 HDACi screened, 13 were identified as preferentially active in MYC-driven MB, compared with the other brain tumor models.
Results: Tacedinaline is a selective inhibitor for MYC-driven medulloblastoma
The study found that CI-994 (tacedinaline), a class I specific HDACi, was the most selectively active compound for MYC-driven MB, with a mean IC50 of 5.97 µM.
CI-994 induces apoptosis and decreases MYC expression in MYC-driven MB cell lines
Study authors determined the induction of apoptosis after HDACi treatment in D425 MED and MED8A, two well-characterized MYC-amplified MB models, to elucidate the antitumoral effect of CI-994 on MYC-driven MB.
CI-994 inhibits growth and reduces leptomeningeal dissemination of MYC-driven MB in vivo
Study authors tested the efficacy of CI-994 in two orthotopic xenograft mouse models of MYC-driven MB in vivo. They found CI-994 significantly:
- Decreased tumor growth in both mouse models
- Prolonged median survival after tumor engraftment compared to control mice
- Control mice
- 34 days for MED8A
- 15 days for D425 MED
- Mice receiving CI-994 treatment
- 31 days for MED8A
- 23 days for D425 ME
- Control mice
- Reduced spinal dissemination in the CI-994 treatment group in both MYC-driven models
These findings demonstrated antitumoral activity of CI-994 against the primary site and, importantly, against the metastatic compartment of MYC-driven MB in vivo. They also identified a novel therapeutic agent targeting the metastatic compartment.
CI-994 induces NF-κB pathway activation in MYC-driven MB
CI-994 also showed significant cell viability reduction mediated by induction of apoptosis in MYC-driven MB.
The study authors assessed the molecular effects of CI-994 on transcription by performing RNA sequencing on cells treated with CI-994 and vehicle control. They found CI-994 caused:
- Global gene expression changes
- Significantly dysregulated canonical pathways and upstream regulators
- NF-κB pathway activation consistently identified
- Most upregulation in target gene transglutaminase 2 (TGM2)
- Significant upregulation of TGM2 mRNA and protein levels in D425 MED and MED8A cells
CI-994 induces tumor inflammation in MYC-MB cell lines and increases infiltration of inflammatory macrophages in vivo
The study authors investigated the role of TGM2 upregulation by CI-994 in tumor inflammation. Treatment with CI-994 (5 μM) alone significantly increased secretion of CCL2/MCP-1, IL-1β and TNF-α. Secretions were suppressed by TGM2 inhibitors ZDON and ERW1041E.
The study authors then investigated if CI-994 treatment of MYC-MB xenograft tumor-bearing mice induced macrophage infiltration. They found that mice treated with CI-994 had increased infiltration of macrophages, and compared to untreated mice, there was an increased number of macrophages with M1 phenotype markers (CD80+CD64+) in xenografts from D425 MED.
CI-994 enhances anti-CD47 mediated phagocytosis and survival of MYC-MB tumor-bearing mice
Study authors investigated the effect of CI-994 mediated NF-kB activation on macrophage checkpoint immunotherapy. Calreticulin was expressed on the surface of MYC-driven MB cells, and its cell-surface exposure was upregulated by CI-994 treatment. There was a significant increase in release of high mobility group box 1 protein, a DAMP protein, in CI-994 treated groups.
Findings include:
- CI-994 combined with anti-CD47 monoclonal antibody (mAb) strongly enhanced phagocytosis of D425 cells by human PBMC-derived macrophages.
- Only a slight increase in phagocytosis from the treatment of anti-CD47
- When results were validated by in vivo experiments in two different MYC-MB orthotopic xenograft models, they showed the combination treatment of CI-994 and anti-CD47 significantly enhanced survival, compared with CD47 or CI-994 alone.
Discussion and conclusion: Tacedinaline and anti-CD47 fight MYC-amplified medulloblastoma
Findings from this study suggest a dynamic relationship between MYC amplification and innate immune suppression in MYC-driven MB and support further investigation of phagocytosis modulation as a strategy to enhance cancer immunotherapy responses.
The combination of anti-CD47 and HDACi eliminated MYC-driven cancer cells with increased efficacy due to the enhancement of the NF-kB mediated “eat-me” signals and antibody-dependent block of “don’t eat-me” signals.
A future clinical trial of tacedinaline and anti-CD47 is forthcoming to determine this treatment's short- and long-term effects.
Featured Researchers
Siddhartha Mitra, PhD
Assistant professor
Pediatrics-Hemetology/Oncology and Bone Marrow Transplantation
University of Colorado School of Medicine
Sujatha Venkataraman, PhD
Associate research professor
Pediatrics-Hematology/Oncology and Bone Marrow Transplantation
University of Colorado School of Medicine
Rajeev Vibhakar, MD, PhD, MPH/MSPH
Pediatric oncologist
Center for Cancer and Blood Disorders
Children's Hospital Colorado
Professor
Pediatrics - Hematology/Oncology and Bone Marrow Transplantation
University of Colorado School of Medicine
