Mayo Clinic

Identifying Novel Molecular Targets for Developing Therapies for Brain Tumor Gioblastoma.

Major Goals of the Project

The goals of this work supported by “Humor to Fight the Tumor” are first, to identify novel druggable modulators of TMZ sensitivity. For this, we proposed to conduct screening using siRNA targeting druggable candidates that were identified through a prior whole genome shRNA library screening. We planned to array the siRNA in 96–well plates and monitored growth of cells expressing these siRNA in an Incucyte culture machine. We proposed to conduct this screening with and without temozolomide (TMZ). We anticipated that this process would identify targets that can slow the growth of GBM cells alone and others in combination with TMZ. The second goal was to identify inhibitor(s) with optimum sensitization of TMZ in GBM. Since the siRNA screening included candidates with available pharmacologic inhibitors, this study was planned to identify candidates targeted by inhibitors with the highest negative impact on the growth of GBM cells alone and together with TMZ. The corresponding inhibitors are considered highly relevant and will be further evaluated in vitro across multiple GBM cells. Overall, this investigation is aimed to complement work supported by a recently awarded NIH R01 and to enhance competitiveness for future NIH awards advancing this important research for GBM patients.

To date, Mayo Clinic has finished the initial screening in GBM43 and U251 cells using siRNA targeting 80 candidates. These candidates were selected out of 603 potential druggable candidates based on whether they control cellular functions that can easily be targeted by pharmacologic inhibitors. Based on this criterion, we initially screened genes mainly involved in cellular signaling, including kinases, phosphatases, receptors, growth factors and other enzymes. The analysis of this data is still in progress and we believe that this screening will yield intriguing findings. During our initial evaluation, we found that siRNA targeting ribonucleotide reductase regulatory subunit M2 (RRM2), pre-mRNA-processing factor 19 (PRPF19), N-alpha-acetyltransferase 50 (NAA50), and leukotriene B4 receptor (LTB4R) suppress growth of glioma cells either alone and with TMZ (Figure 1 and data not shown). Since O6-methylguanine-DNA-methyltransferase (MGMT) is critical for TMZ sensitivity, we evaluated the effect siRNA targeting MGMT as a positive control. As expected, MGMT siRNA sensitized MGMT-expressing GBM43 cells to TMZ (Figure 2), suggesting that our siRNA screening worked faithfully.

Future Plans to Accomplish the Goals

We are planning to continue evaluating the candidates we identified by the current secondary screening. For this, we will use siRNA to target these candidates in 10 (5 MGMT+ and 5 MGMT-) patient derived GBM xenografts. We are anticipating that by conducting additional screening we will eventually identify 2-3 best candidates for the next evaluation using shRNA approach. These shRNA experiments will be performed both in vitro and in vivo. In a situation whereby pharmacologic inhibitors are available, these inhibitors will be used to target the candidates instead of shRNA, especially if these inhibitors can penetrate the brain. In parallel, with the above described work, we are planning to continue screening beyond the initial 80 candidates. Our goal is to identify candidates that can be used to generate mechanistic hypotheses for applying NIH support through R01 award. We thank Humor to Fight Tumor for supporting this work and we are optimistic that this research will identify novel targets for developing therapy for patients with GBM, and perhaps other human cancers.

Figure 1: Representative example of siRNA target (NAA50) that suppresses growth of GBM cells alone and with TMZ.

Figure 2: Sensitization of MGMT expressing GBM43 cells to TMZ by siRNA targeting MGMT, which was used as a positive control. TMZ 100 microM was given 24 hours after siRNA transfection and growth was monitored in Incucyte machine.