A new approach in preclinical drug testing for the treatment of GBM

Glioblastoma multiforme (GBM) is the most aggressive form of primary brain cancer that affects adults. It is a devastating disease with an extremely poor prognosis, with a median survival of 14 to 16 months after treatment.1

The current standard of care, surgical resection followed by concomitant administration of radiation and temozolomide (Temodar), has remained unchanged for nearly 2 decades.2.3 The FDA has not approved a drug for GBM since temozolomide, and no new agent has been shown to prolong patient survival.

One of the underlying reasons for the clinical failure of promising therapies in GBM is inadequate testing of new treatments at the preclinical level. The Ivy Brain Tumor Center at the Barrow Neurological Institute in Phoenix, Arizona is trying to subvert this pattern by changing the way preclinical studies are designed and conducted to assess the effectiveness of GBM drugs.

One of the biggest challenges in the field of brain cancer treatment is that most drugs cannot cross the blood-brain barrier (BBB), which prevents the passage of potentially harmful substances, including drugs, into the brain tissue.4.5 As these drugs cannot enter the brain, they do not accumulate in GBM tissue in therapeutically relevant concentrations, resulting in clinical failure..6

Although evaluation of drug pharmacokinetics (PK) in clinical trials of GBM is becoming more common, the PK properties of most drugs are not routinely evaluated in preclinical models of GBM. However, all new drugs tested at the Ivy Brain Tumor Center are extensively scrutinized for 2 key parameters: is the drug accumulating to sufficient concentrations within the tumor tissue and, if so, is the drug exerting its intended pharmacodynamic (PD) effects on the tumor cells? We used this extensive PK-PD pairwise analysis approach to conclusively determine the translational potential of any drug in preclinical models of GBM. Our goal is to accelerate the translation of encouraging preclinical findings into new therapies for patients.

In a preprint available on the BioRxiv server, we report a comprehensive PK-PD correlation analysis in preclinical GBM models for the second-generation HDAC inhibitor quisinostat.7 Quisinostat was chosen as a promising agent because it has high specificity for HDAC1, a protein that we previously demonstrated to be essential for the survival of cancer stem cells present in GBM.

Figure.  Chisinostat, a Brain-Penetrating HDACi, Sensitizes GBM Cells to Radiation Treatment

Figure. Chisinostat, a Brain-Penetrating HDACi, Sensitizes GBM Cells to Radiation Treatment

We first conducted preliminary in vitro studies to examine the efficacy of kisino-stat against patient-derived GBM cell lines and found that the agent potently reduced GBM cell viability at low nanomolar concentrations. Interestingly, when we examined the molecular consequences of quisinostat treatment in more detail, we found that short-term incubation with the drug resulted in extensive accumulation of DNA damage in GBM cells. The longer the incubation period with quisinostat, the greater the amount of DNA damage in the cancer cells.

As quisinostat acts as a DNA damaging agent, we next questioned whether it could enhance the effects of radiation, which is the mainstay of GBM treatment.3 In other solid tumors, there is evidence that HDAC inhibitors can sensitize cancer cells.8-12 We hypothesize that the accumulation of quisinostat-induced DNA damage in combination with radiation treatment would synergistically reduce the viability of GBM cells. In fact, we found that quisinostat synergized robustly with radiation treatment in several GBM cell lines.

The killer experiment was to determine whether Quisinostat could cross an intact BBB. To answer this question, we treated mice with Quisinostat on a short-term basis and subsequently collected their plasma and brain for corresponding PK and PD analyses.

It is worth noting that although pharmacokinetic analyzes are sometimes performed in preclinical trials for a variety of different brain tumors, these studies generally measure only the total brain to plasma concentration ratio as a measure of drug penetration into the brain. brain.13-15 However, the value of this relationship is quite limited and may lead to erroneous conclusions, as it does not consider the protein or lipid binding fraction of the drug in plasma and brain.16

We employ a method called equilibrium dialysis to measure unbound, or “free,” drug concentrations, which represent the pharmacologically active fraction of a drug. We found that although total levels of quisinostat were abundant in the brain, only a very small fraction of the drug did not bind. However, these low concentrations were sufficient to induce robust target modulation (PD response) in brain tissue.

After establishing that Kisinostat is indeed a BBB-penetrating drug, we subsequently evaluated its efficacy by treating tumor-bearing mice with Quisi-Nostat, radiation, or a therapy combining the two. Although quisinostat alone did not provide a survival benefit, when combined with radiation, all animals lived significantly longer compared to radiation monotherapy. To our knowledge, this is the first report demonstrating that quisinostat can act as a potent radiosensitizer in any preclinical model of cancer.

Interestingly, RNA sequencing analysis of the resulting tumors revealed that the combination therapy caused tumor cells to adopt neuronal-like cellular fates, increased levels of oxidative stress, and decreased expression of genes involved in DNA repair and cell division. . This suggests that concomitant treatment with quisinostat and radiation may induce transcriptional changes that promote cell differentiation, cell cycle arrest, and cell death.

The identification of drugs that potentiate the effects of radiation treatment is an intense area of ​​research in neuro-oncology. Here, we provide the first report that quisinostat is a brain-penetrating HDAC inhibitor with potent radiosensitization properties in preclinical GBM models. Our findings also have important implications for the management of other malignancies – such as prostate, colon, lung and esophageal cancers – where HDACs are often overexpressed and where radiation is commonly used as a treatment modality.17.18

A new approach in preclinical drug testing for the treatment of GBM

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