Two groups of participants committed the same mock crime in which one of two items, a watch or a ring, was removed from a drawer and concealed. One group, the crime‐familiar group next experienced a three‐stimulus protocol (3SP), a Concealed Information Test (CIT), in which they were tested on the stolen (probe) item presented in a random series of five irrelevant (unseen) stimuli from the same jewelry category. A left‐hand button press, meaning “I don't recognize” was to follow each of these six items. A right‐hand press (“I do recognize”) was to follow the one other presented item, the target item, which in the case of the crime‐familiar group was the other, not‐stolen item in the drawer at the mock crime scene. For the other crime‐unfamiliar group, the target was a sixth unseen irrelevant item as in the original P300 CIT. In terms of P300 latency and reaction time (RT), crime‐familiar participants processed all stimuli faster than crime‐unfamiliar participants. The CIT effects (probe‐minus‐irrelevant differences) for crime‐familiar group members were inferior to those of crime‐unfamiliar group members for RT and P300 amplitude measures. Thus, familiar targets negatively impact the 3SP.
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<0.0001). 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<0.01), 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.
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.
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.
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.
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