(M1430-04-23) Combination Therapy of BRD4/PI3K Dual Inhibitor and a Novel Hedgehog Inhibitor for the Treatment of Sonic Hedgehog and MYC-Amplified Medulloblastoma

Purpose: Medulloblastoma (MB) is a major pediatric malignant brain tumor that arises from the cerebellum. Among the four subgroups of MB, Sonic Hedgehog (SHH) and Group 3 MB represent most of the MB cases. Aberrant activation of the SHH pathway promotes the proliferation of chemo-resistant cancer stem cells (CSCs) and Group 3 MB is characterized by upregulation of the MYC oncogene which lies downstream of the phosphoinositide 3-kinase (PI3K) signaling pathway. To address this therapeutic need, we have identified SRX3305 a potent multi-action inhibitor of PI3K and bromodomain-containing 4 (BRD4)-MYC that yields significant anti-tumor effects and spares healthy cells(1). SMO mutation is responsible for resistance in SHH MB due to downstream GLI (glioma-associated oncogene) activation. As a result, our lab has developed MDB5, a novel inhibitor of the smoothened (SMO) gene in the SHH pathway and we have shown previously its binding to SMO mutants as confirmed by the molecular docking studies(2). Also, it has been observed that overexpression and activation of non-canonical pathways like PI3K signaling frequently occur in SHH-MB resistant to SMO inhibitors. This crosstalk of SHH with PI3K pathway is associated with high-risk SHH and Group 3 MB. Thus, simultaneous inhibition of the PI3K and SHH pathway to suppress MYC and GLI expression has the potential to irreversibly diminish MB. We have found that a combination treatment of MDB5 and SRX3305 in MB cells can synergistically inhibit cell proliferation. Also, SRX3305 showed a six-fold reduction in IC₅₀ value as compared to another PI3K-BRD4 inhibitor SF2523 in SHH amplified DAOY cells which we have used in our previous study (3). This suggests a significantly higher potency of SRX3305 compared to SF2523. Hence, we propose that a combination treatment of SRX3305 and MDB5 can promote effective therapy for treating chemoresistance in MB.

Methods: We performed in-vitro cell cytotoxicity assay to evaluate the efficacy of SF2523 against SRX3305 and for the combination treatment of SRX3305 and MDB5 in SHH-amplified DAOY cells and MYC-amplified HDMB03 cells. For combination treatment, the concentration of one drug was varied and the other was kept constant. The cytotoxic effect of individual drugs and combination treatment was determined using the MTT assay and the IC₅₀ values and the combination index was measured using Compusyn software. Next, the apoptotic activity of MDB5, SRX3305 and their combination was analyzed in DAOY cells.  Since, GLI proteins play a crucial role in tumor onset, progression, metastasis and cancer stem cell maintenance, we checked the effect of individual drugs and their combination on GLI expression. For the GLI inhibition assay we treated DAOY cells with SRX3305 and MDB5 at their IC₅₀ dose and for the combination treatment we used both the drugs a concentration of their IC₂₅ for a period of 24 hours. Further, we extracted the proteins from these cells and checked the protein expression by western blot. Since, the efficacy of SRX3305 has not been evaluated in vivo, we checked the stability of SRX3305 in cerebrospinal fluid as a preliminary analysis.

Results: The individual IC₅₀ values for SRX3305 were found to be 3.96 µM and 2.45 µM, for SF2523 were found to be 13.49 µM and 14.49 µM and for MDB5 were found to be 114 µM and 150 µM respectively in HDMB03 and DAOY cells. This suggests that there was a 5.8-fold and 3.4-fold reduction in IC₅₀ for SRX3305 as compared to SF2523 in DAOY and HDMB03 cells respectively suggesting the higher potency of SRX3305 over SF2523. Also, a combination of SRX3305 and MDB5 reduced the IC₅₀ to 0.57µM and 0.2µM in HDMB03 and DAOY cells respectively. The Combination index (CI) value was less than 1 indicating synergism between the two drugs. We also observed an increase in the percentage of early and late apoptotic cells in combination treatment (40.4%) as compared to SRX3305 (17.7%), MDB5 (6.5 %) treated and the control cells (3.5%). We also found that a combination treatment of SRX3305 and MDB5 in DAOY cells significantly reduced expression of GLI1 as compared to individual drugs and control as shown by analysis of protein expression using western blot. Also, SRX3305 was stable in the cerebrospinal fluid for 72 hours with only 28% of drug degradation.

Conclusion: As per the results, SRX3305 proved to be significantly more potent than our previously used BRD4-PI3K inhibitor SF2523. Also, the combination of SRX3305 along with MDB5 displayed synergism in vitro as indicated by the cell cytotoxicity, cell apoptosis and GLI inhibition assay. SRX3305 also displayed stability in-vitro in the cerebrospinal fluid. Thus, owing to the synergism displayed by both SRX3305 and MDB5 in vitro we conclude that the combination of both these drugs would be an effective therapy in the treatment of chemoresistant MB.

References: 1. Vann KR, Pal D, Smith AL, Sahar N e., Krishnaiah M, El-Gamal D, et al. Combinatorial inhibition of BTK, PI3K-AKT and BRD4-MYC as a strategy for treatment of mantle cell lymphoma. Molecular Biomedicine. 2022 Dec 1;3(1).
2. Kumar V, Chaudhary AK, Dong Y, Zhong HA, Mondal G, Lin F, et al. Design, Synthesis and Biological Evaluation of novel Hedgehog Inhibitors for treating Pancreatic Cancer. Sci Rep. 2017 Dec 1;7(1).
3. Kumar V, Wang Q, Sethi B, Lin F, Kumar V, Coulter DW, et al. Polymeric nanomedicine for overcoming resistance mechanisms in hedgehog and Myc-amplified medulloblastoma. Biomaterials. 2021 Nov 1;278.

Acknowledgements: The NIH (1R01NS116037), pediatric Cancer Research Group of the University of Nebraska Medical Center and Children’s Hospital (LB805), Team Jack Foundation, Nebraska DHHS LB506, Nebraska Research Initiative (NRI) Program, and the Faculty Start-up fund to Ram Mahato are duly acknowledged for providing financial support for this work.


Figure 1. Proposed mechanism of action for SRX3305 and MDB5


Figure 2: Comparison of cell cytotoxicity of SRX3305 and SF2523 in A) HDMB03 cells B) DAOY cells.
Cell cytotoxicity and combination index plot for SRX3305 and MDB5 in C) HDMB03 cells D) DAOY cells


Figure 3. A) Induction of apoptosis by SRX3305 and MDB5 in DAOY cells
B) GLI-1 inhibition after combination treatment of SRX3305 and MDB5 in DAOY cells