Dual Targeting of PI3K and HDAC by CUDC-907 Inhibits Pediatric Neuroblastoma Growth

Abstract

The dysregulation of PI3K, HDACs, and MYCN are well known for promoting multiple cancer types, including neuroblastoma (NB). Targeting the upstream regulators of MYCN, including HDACs and PI3K, was shown to suppress cancer growth. In the present study, we analyze different NB patient datasets to reveal that high PI3K and HDAC expression is correlated with overall poor NB patient survival. High PI3K level is also found to be associated with high MYCN level and NB stage progression. We repurpose a dual inhibitor CUDC-907 as a single agent to directly target both PI3K and HDAC in NB. We use in vitro methodologies to determine the efficacy and selectivity of CUDC-907 using six NB and three control fibroblast cell lines. Our results show that CUDC-907 significantly inhibits NB proliferation and colony growth, induces apoptosis, blocks cell cycle progression, inhibits MYCN, and enhances H3K9Ac levels by inhibiting the PI3K/AKT signaling pathway and HDAC function. Furthermore, CUDC-907 significantly inhibits NB tumor growth in a 3D spheroid tumor model that recapitulates the in vivo tumor growth. Overall, our findings highlight that the dual inhibition of PI3K and HDAC by CUDC-907 is an effective therapeutic strategy for NB and other MYC-dependent cancers.

Inhibition of Polo-like Kinase 1 by HMN-214 Blocks Cell Cycle Progression and Inhibits Neuroblastoma Growth

“…Six human NB cell lines—including both MYCN-amplified (NGP, LAN-5, IMR-32) and MYCN-non-amplified (SH-SY5Y, SK-N-AS, CHLA-255) cell lines and three normal fibroblast control cell lines (WI-38, NIH-3T3, COS-7)—were routinely cultured and maintained as described previously [ 41 ]. All the cell lines used in this study were validated via short-tandem repeat analysis for genotyping within the last six months and were tested for mycoplasma monthly.…”

“…Apoptosis assays were performed using Muse Annexin V & Dead Cell Kit (MCH100105; Luminex Corp, Austin, DX, USA), and cell cycle assays were performed using Click-iT™ Plus EdU Alexa Fluor™ 488 Flow Cytometry Assay Kit (C10633; ThermoFisher Scientific, Waltham, MA, USA) and FxCycle™ PI/RNase Staining Solution (F10797; ThermoFisher Scientific) according to the manufacturer’s instructions and as described previously [ 41 ]. Briefly, 1 × 10 6 NB cells per well were seeded in a six-well plate followed by treatment with different doses of HMN-214 for 16 h. Apoptosis assays were performed using Muse flow cytometer (Luminex) and cell cycle assays were performed using Attune Nxt Flow Cytometer (ThermoFisher Scientific).…”

“…Three-dimensional spheroidal 96-well microplates (4515; Corning, NY, USA) were used to perform 3D spheroidal tumor assays as described previously [ 41 ]. Briefly, 300 µm sized spheroids were randomly selected and treated with different concentrations of HMN-214.…”

“…Gene expression analysis was performed using the RT-qPCR analysis as described previously [ 41 ]. Briefly, 1 × 10 6 NB cells per well were seeded into a 6-well plate and treated with the different concentrations of HMN-214 for 12 h. Total RNA was extracted using RNeasy plus mini kit (74134; Qiagen) according to manufactures instructions and as described previously [ 41 ]. High-capacity cDNA reverse transcription kit (4368814; ThermoFisher Scientific) was used to synthesize cDNA from total RNA according to manufactures instructions.…”

“…High-capacity cDNA reverse transcription kit (4368814; ThermoFisher Scientific) was used to synthesize cDNA from total RNA according to manufactures instructions. Further, RT-qPCR reactions for individual genes were performed using SYBR Green dye (4385610; ThermoFisher Scientific) using the QuantStudio 3 Real-Time PCR System (ThermoFisher Scientific) as described previously [ 41 ]. Primers were designed using the NCBI primer design software and listed in Table 1 .…”