Search

During therapy, adaptations driven by cellular plasticity are partly responsible for driving the inevitable recurrence of glioblastoma (GBM). To investigate plasticity-induced adaptation during standard-of-care chemotherapy temozolomide (TMZ), we performed in vivo single-cell RNA sequencing in patient-derived xenograft (PDX) tumors of GBM before, during, and after therapy. Comparing single-cell transcriptomic patterns identified distinct cellular populations present during TMZ therapy. Of interest was the increased expression of ribonucleotide reductase regulatory subunit M2 ( RRM2 ), which we found to regulate dGTP and dCTP production vital for DNA damage response during TMZ therapy. Furthermore, multidimensional modeling of spatially resolved transcriptomic and metabolomic analysis in patients’ tissues revealed strong correlations between RRM2 and dGTP. This supports our data that RRM2 regulates the demand for specific dNTPs during therapy. In addition, treatment with the RRM2 inhibitor 3-AP (Triapine) enhances the efficacy of TMZ therapy in PDX models. We present a previously unidentified understanding of chemoresistance through critical RRM2-mediated nucleotide production.

Ten novel mitochondria-targeted dihydroartemisinin ether derivatives were designed, synthesized, and evaluated for antitumor activity against five cancer cell lines in vitro. Profoundly, compound

Glioblastoma (GBM) remains one of the most resistant and fatal forms of cancer. Previous studies have examined primary and recurrent GBM tumors, but it is difficult to study tumor evolution during therapy where resistance develops. To investigate this, we performed an in vivo single-cell RNA sequencing screen in a patient-derived xenograft (PDX) model. Primary GBM was modeled by mice treated with DMSO control, recurrent GBM was modeled by mice treated with temozolomide (TMZ), and during therapy GBM was modeled by mice euthanized after two of five TMZ treatments. Our analysis revealed the cellular population present during therapy to be distinct from primary and recurrent GBM. We found the Ribonucleotide Reductase gene family to exhibit a unique signature in our data due to an observed subunit switch to favor RRM2 during therapy. GBM cells were shown to rely on RRM2 during therapy causing RRM2-knockdown (KD) cells to be TMZ-sensitive. Using targeted metabolomics, we found RRM2-KDs to produce less dGTP and dCTP than control cells in response to TMZ (p). Supplementing RRM2-KDs with deoxycytidine and deoxyguanosine rescued TMZ-sensitivity, suggesting an RRM2-driven mechanism of chemoresistance, established by regulating the production of these nucleotides. In vivo, tumor-bearing mice treated with the RRM2-inhibitor, Triapine, in combination with TMZ, survived longer than mice treated with TMZ alone (p), indicating promising clinical opportunities in targeting RRM2. Our data present a novel understanding of RRM2 activity, and its alteration during therapeutic stress as response to TMZ-induced DNA damage.

Glioblastoma (GBM) is an aggressive primary brain tumor characterized by poor prognostic outcomes. Nearly all GBM tumors recur, due to cellular resistance against chemotherapies, including temozolomide (TMZ), which develops during active treatment. To investigate these adaptive mechanisms, our lab conducted a single-cell RNA sequencing analysis utilizing an in vivo patient-derived xenograft (PDX) model of GBM, prior to, during, and post TMZ-based therapy. Our data revealed 149 genes to be uniquely expressed in the during-TMZ condition (p < 0.0001), including two regulatory enzymes involved in purine metabolism: the Ribonucleotide Reductase (RNR) Regulatory Subunit 2, RRM2, and inosine monophosphate dehydrogenase 2, IMPDH2. Previous work in this lab has independently established both RRM2 and IMPDH2 as significant drivers of TMZ-resistance in GBM, through their respective abilities to alter tumor metabolism during therapy. Network analysis of genetic pathways enriched during TMZ therapy revealed a previously unidentified interaction between IMPDH2 and the RRM1 subunit of the RNR enzyme. Given these data, we aimed to determine if there was also an interaction between IMPDH2 and RRM2 in GBM. Immunocyto- and histochemistry of GBM-PDX primarily demonstrated significant co-localization between RRM2 and IMPDH2 during TMZ therapy. Immunoprecipitation (IP) and reverse-IP analyses were subsequently conducted, and revealed a previously unreported molecular interaction between the two proteins in GBM lines, which increased in a TMZ-dependent manner. These results were not corroborated in neural stem cell lines. Additionally, immunoblot analyses revealed that in RRM2-knockdown GBM-PDX, IMPDH2 protein expression is decreased compared to controls. We hypothesize the dynamic interaction of RRM2 and IMPDH2 to enhance the metabolic adaptations underlying GBM chemoresistance. The efforts of this study are now focused on investigating the potentially synergistic mechanisms of FDA-approved RRM2 and IMPDH2 inhibitors, previously shown to enhance the efficiency of TMZ. This novel and targetable interaction may present a promising clinical opportunity for GBM therapy.

Background Bladder cancer is the second most common genitourinary malignancy and the eleventh most common cancer worldwide. Dihydroartemisinin (DHA), a first-line antimalarial drug, has been found to have potent antitumor activity. In our previous study, a novel dihydroartemisinin derivative Mito-DHA5 synthesized in our laboratory has a stronger anti-tumor activity than DHA. In this study, we investigated the apoptotic effect of Mito-DHA5 on bladder cancer T24 cells and molecular mechanisms underlying. Methods Antitumor activity in vitro was evaluated by MTT, wound healing and cloning formation assays. Mitochondrial membrane potential (MMP) was detected by JC-1 probe and ROS levels were measured by specific kit. The expression of caspase-3, cleaved-caspase3, mitochondrial Cyt-C, Bcl-2, Bax and PARP in T24 cells was evaluated by Western blotting. Results The results showed that Mito-DHA5 reduced cell viability with an IC50 value of 3.2 µM and induced T24 cell apoptosis in a dose-dependent manner, increased the production of ROS and decreased MMP. Mito-DHA5 could down-regulate the expression of Bcl-2, mitochondrial Cyt-C, Caspase-3, PARP and up-regulate the expression of Bax and cleaved Caspase-3. Conclusions These data suggested that Mito-DHA5 had a potent inhibitory effect on T24 bladder cancer cell growth and induced these cells apoptosis associated with mitochondrial pathway.

Glioblastoma (GBM) remains one of the most resistant and fatal forms of cancer. Previous studies examine pre- and post-tumor recurrence; however, it is incredibly difficult to study tumor evolution during therapy where resistance develops. To investigate this, our lab performed a single-cell RNA-sequencing screen before, during, and after temozolomide-based (TMZ) chemotherapy in a patient-derived xenograft (PDX) model in vivo. Our analysis found 149 genes uniquely expressed during TMZ-therapy compared to pre- and post-therapy (p< 0.0001). Of these, the ribonucleotide reductase (RNR) gene family stood out due to the preferential switch to Ribonucleotide Reductase Regulatory Subunit M2 (RRM2) during therapy. Classically, RRM2, or its isoform RRM2B, forms a complex with RRM1 to create an RNR, mediating deoxynucleoside triphosphate (dNTP) production. Our single-cell data revealed that GBM cells rely on RRM1-RRM2 interaction during therapy, but switch to RRM1-RRM2B in post-therapy recurrent GBM. In vitro, RRM2-knockdown cells increased TMZ susceptibility, whereas RRM1- and RRM2B-knockdowns were more resistant to TMZ (p< 0.001). Immunocytochemistry found elevated yH2AX fluorescence in RRM2-knockdowns after TMZ treatment, signifying reduced DNA repair capacity compared to the control (p< 0.001). To understand the mechanism of RRM2-mediated chemoresistance, targeted metabolomics was applied to quantify dNTP signatures during TMZ-therapy. In response to TMZ, dCTP and dGTP production in GBM cells increased 100-fold and 80-fold respectively (p< 0.001). RRM2-knockdowns produced significantly less dCTP and dGTP (p< 0.0001). By supplementing RRM2-knockdowns with dCTP and dGTP, TMZ-susceptibility was rescued, suggesting that RRM2 drives chemoresistance by promoting production of these two nucleotides. In vivo, following intracranial injection of GBM cells, mice treated with the RRM2 inhibitor Triapine with TMZ survived longer than those treated with TMZ alone, indicating promising clinical opportunities in targeting RRM2 (p< 0.0001). Overall, our data present a novel understanding of how RRM2 activity is altered during therapeutic stress to counteract TMZ-induced DNA damage.

Glioblastoma (GBM) is the most common adult malignant brain tumor. Despite an aggressive standard of care involving surgical resection, radiation therapy, and temozolomide (TMZ)-based chemotherapy, GBM remains a fatal disease due to a 100% recurrence rate. Tumor heterogeneity and cell plasticity are the main contributors to GBM recurrence after treatment, and thus it is essential to study the mechanisms of therapy-induced cellular plasticity. To identify these mechanisms, we performed in vivo single-cell analysis of primary (no therapy), during therapy, and post-therapy recurrent model patient-derived xenograft model of GBM. The analysis revealed a higher transcription of tryptophanyl-tRNA synthetase (trpRS) during TMZ therapy (p < 0.0001). Across multiple datasets, elevated trpRS was shown to correlate significantly with shorter GBM patient survival times (p = 0.0069). TrpRS has been noted to have non-canonical functions, including the ability to be secreted and act as a cytokine in response to injury, often to upregulate stemness pathways to promote healing and regeneration. Based on this observation, we hypothesized that during TMZ treatment, trpRS secretion promotes stemness in neighboring GBM cells via activation of the phosphatidylinositol-3-kinase/protein kinase B (PI3K/Akt) pathway. This study shows that in response to TMZ treatment, secreted trpRS levels are significantly elevated compared to control in multiple GBM cell lines as analyzed by western blotting. Additionally, expression of trpRS is elevated in TMZ-resistant GBM6R cells compared to TMZ-sensitive GBM6. Furthermore, phosphorylated Akt levels, indicative of the activation of the pro-stemness PI3K/Akt pathway, correlate positively with trpRS. This study, one of the few to examine genetic changes during therapy, sheds light on a novel therapeutic target for GBM.

Copy move forgery detection (CMFD) is one of the most active subtopic in forgery scheme. The methods of CMFD are divided into to block-based method and keypoint-based method in general. Compared with keypoint-based method, block-based method can detect undetectable detail without morphology segmentation. But many block-based methods detect the plain copy-move forgeries only. They have been incompetent to detect the post-processing operations such as various geometrical distortions, and then fail to detect the forgery regions accurately. Therefore, this paper presents an improved block-based efficient method for CMFD. Firstly, after pre-processing, an auxiliary overlapped circular block is presented to divide the forged image into overlapped circular blocks. The local and inner image feature is extracted by the Discrete Radial Harmonic Fourier Moments (DRHFMs) with the overlapped circular block from the suspicious image. Then, the similar feature vectors of blocks are searched by 2 Nearest Neighbors (2NN) test. Euclidean distance and correlation coefficient is employed to filter these features and then remove the false matches. Morphologic operation is employed to delete the isolated pixels. A series of experiments are done to analyze the performance for CMFD. Experimental results show that the new DRHFMs can obtain outstanding performance even under image geometrical distortions.

Copy move forgery with geometric distortions such as the rotational operation, the scaling operation, the mirror operation and the additive noise operation became more common. Existing methods are not competent for the detection of the copy move forgery with these distortions. In fact, the most critical issue for the detection of the forgery is the determination of the geometric features. This paper proposes an efficient Discrete Radon Polar Complex Exponential Transform (DRPCET)-based method for the extraction of the rotational and the scaling invariant features for the copy move forgery detection. First, the features obtained by the Radon transform (RT) and the Polar Complex Exponential Transform (PCET) are fused together. Then, these features are normalized. In order to achieve the scaling invariant property, an auxiliary circular template is introduced. With the auxiliary circular template, the translational moment invariant features, the rotational moment invariant features and the scaling moment invariant features are constructed for the extraction of the planar geometrical features. By further extracting some useful features for the representation of the image background, the interference of the background information can be reduced. After extracting the geometrical features, the lexicographic sorting is applied. Then, a correlation between the same part or similar parts of the image which are copied and moved to another image is computed. Based on the obtained correlations, these forgery parts can be identified and their composed positions can be located. Finally, these images are denoted as the forgery image. Extensive computer numerical simulations have been performed. The obtained results show that the proposed method can detect the copy move region in the forgery image precisely even though the forgery regions are suffered from the mixed geometric distortions.