Group 3 affiliation and MYC genetic amplification are associated with poor life expectancy and substantial morbidity in children suffering from medulloblastoma (MB). However, the high metabolic demand induced by MYC-driven transformation sensitizes MYC-overexpressing MB to cell death under conditions of nutrient deprivation (ND). Additionally, MYC-driven transformation is known to promote mitochondrial oxidative phosphorylation (OXPHOS). We previously reported that eukaryotic Elongation Factor Kinase 2 (eEF2K), the master regulator of mRNA translation elongation, promotes survival of MYC-overexpressing tumors under ND. Interestingly, eEF2K is overexpressed in MYC-driven MB and our preliminary proteomics data highlight large-scale alterations in OXPHOS components affecting eEF2K deficient MB cells. We therefore hypothesized that eEF2K activity is required for the selective translation of mRNAs needed for efficient OXPHOS, and for the progression of MYC-driven MB. We performed Multiplexed enhanced Protein Dynamic Mass Spectrometry in eEF2K knockdown MYC-overexpressing D425 MB cells to identify mRNAs selectively translated upon eEF2K activation. Messenger RNAs encoding multiple (9 out of 10 detected) components of the mitochondrial OXPHOS pathway are selectively translated upon eEF2K activation. Inactivation of eEF2K by genetic KO leads to the disassembly of electron transport chain (ETC) complexes I-IV without affecting mRNA levels of their respective components. Consistently, eEF2K KO MB cells display decreased mitochondrial membrane potential and 20% increased proton leak thorough the mitochondrial membrane. In addition, eEF2K inactivation results in increased Group 3 MB cell death under ND and doubles survival of MB bearing mice fed with calorie restricted diets (p< 0.05). Control of mRNA translation elongation by eEF2K is critical for mitochondrial ETC complex assembly and efficient OXPHOS in MYC-overexpressing MB, likely representing an adaptive response by which MYC-driven MB cells cope with acute metabolic stress. Future therapeutic studies will aim to combine eEF2K inhibition with caloric restriction mimetic drugs as eEF2K activity appears critical under metabolic stress conditions.
Background Mycoplasma hominis infection is common in urinary tract. 18F-FDG-PET/CT is a valuable tool for tumor and infection diagnosis. Few studies have shown the 18F-FDG-PET/CT images after mycoplasma infection. Case presentation Here we described a case of Waldenstrom macroglobulinemia with thickened bladder wall. The 18F-FDG-PET/CT showed the SUVmax up to 36.1 mimicking bladder cancer. The results of histopathological examination and metagenomic sequencing of the blood and urinary revealed the Mycoplasma hominis infection. Conclusion The full consideration should be given to the possibility of infection besides tumor in lesions with high SUV value in 18F-FDG-PET/CT, especially in immunodeficiency patients.
Medulloblastoma (MB) is the most common pediatric intracranial tumor and leading cause of childhood related cancer deaths. Group 3 affiliation and genetic amplifications of the MYC oncogene are predictors of adverse outcome in MB, underscoring a dire need for novel and more effective therapeutic approaches. The let-7 family of small non-coding RNAs (miRNAs) is known to inhibit tumor progression and regulate metabolism by targeting and degrading several cellular mRNAs, including MYC. Indeed, let-7 miRNAs are frequently repressed in several cancer types, including in MYC-driven MB. We previously reported that the mRNA translation elongation regulator eukaryotic Elongation Factor-2 Kinase (eEF2K) is a pivotal mediator of cancer cell adaptation to nutrient deprivation. In the current work, we identified a potential binding site for let-7 miRNAs on the eEF2K 3’ untranslated region (UTR). In addition, eEF2K mRNA and let-7 miRNA expressions negatively correlate in MB, suggesting a potential regulation of the former by the latter. Let-7 miRNAs transfection decreases eEF2K mRNA and protein levels (by ~40–50%). Down-regulation of luciferase activity by let-7 miRNAs is impaired upon mutation of the let-7 binding site on the eEF2K 3’UTR. Inhibition of eEF2K significantly reduces survival of MYC-amplified MB cell lines under nutrient deprivation, altering their mRNA translation rates. Knockout of eEF2K increases survival of MYC-amplified MB xenografts when mice are kept under calorie restricted diets. We conclude that let-7 miRNAs degrade the eEF2K mRNA by binding to its 3’UTR, indicating that let-7 repression in MYC-driven MB is partially responsible for increased eEF2K levels. Moreover, the let-7-eEF2K axis constitutes a critical mechanism for MYC-driven MB adaptation to acute metabolic stress, representing a promising therapeutic target. Future therapeutic studies will aim to combine eEF2K inhibition with caloric restriction mimetic drugs, as eEF2K activity appears critical under metabolic stress conditions.
Background: Effective treatment of metastatic disease remains a major challenge in the improvement of patient outcomes in osteosarcoma (OS). Novel anti-metastatic therapies are needed to treat distant metastases. To this end, the current research evaluates whether targeting the dysregulated mRNA translation machinery in OS can inhibit metastases. Hypothesis: We hypothesize that mRNA translation factors present at an abnormally high abundance in OS cells support the rapid synthesis of cytoprotective proteins that are needed to survive in the oxidative stress-rich microenvironment of the lung. Experimental Approach: Databases of OS cell lines and patient tumor data (A. Sweet-Cordero, UCSF) were queried to identify mRNA translation factors with abnormal transcript levels. A limited drug screen of small molecule inhibitors (SMIs) against identified candidates was carried out to evaluate IC50 values in metastatic OS cells. A candidate inhibitor identified from these data was further characterized for synergy with chemical inducers of oxidative stress (e.g. tert-butylhydroquinone [tBHQ]) that mimics conditions encountered in the lung. Drug combination studies examined 2D and 3D tumor spheroid growth, cellular oxidative stress, and PARP-cleavage, under +/- inhibitor and +/- oxidative stress conditions. Metastatic OS cells were engineered to express an antioxidant response element (ARE)-mCherry fluorescent reporter to directly monitor the antioxidant response by fluorescence microscopy. Polysome profiling was used to assess inhibitor-mediated changes in global mRNA translation. The anti-metastatic activity of the inhibitor was tested in the ex vivo pulmonary metastasis assay (PuMA) and in in vivo metastasis models. Results: From cell and patient sample screening, eIF4A1/2 was identified as being abnormally regulated in metastatic OS cells. The SMI, CR-1-31B, specifically targets eIF4A1/2 and was found to have an IC50 of just 8 nM. CR-1-31B was found to inhibit tumor cell growth in 2D and 3D, increase cellular oxidative stress, and enhance PARP-cleavage, but only under oxidative stress conditions. Western analysis of tBHQ-treated metastatic OS cells with the ARE-mCherry reporter confirmed that the temporal expression of mCherry correlated with the upregulation of Nuclear factor erythroid 2-related factor-2 (Nrf2), a key transcriptional regulator of the antioxidant response. CR-1-31B blunted the upregulation of the antioxidant response in 2D and 3D tumor growth conditions with oxidative stress. CR-1-31B, in a dose-dependent manner, decreased the amount of polysomal mRNAs. CR-1-31B reduced the lung tumor burden in the ex vivo PuMA model, delayed primary tumor growth, and reduced lung metastases in in vivo xenograft OS models. Conclusions: Our data demonstrates that dysregulated mRNA translation is a metastatic vulnerability that can be exploited with SMIs. Altogether, these data support the inhibition of metastatic OS by CR-1-31B, highlighting the potential therapeutic utility of this selective translation inhibitor. Citation Format: Michael M. Lizardo, Christopher Hughes, Yue Zhou Huang, Alberto Delaidelli, Taras Shyp, Haifeng Zhang, Sol Snir Shaool, Poul H. Sorensen. A potent eIF4A1/2 inhibitor CR-1-31B down-modulates the antioxidant stress response in osteosarcoma cells and inhibits in vivo lung metastases [abstract]. In: Proceedings of the AACR Special Conference: Cancer Metastasis; 2022 Nov 14-17; Portland, OR. Philadelphia (PA): AACR; Cancer Res 2022;83(2 Suppl_2):Abstract nr A024.
Background Minimal residual disease (MRD) is closely associated with risk stratification of hematological malignancies. Monitoring the MRD levels of patients during treatment and at time points after remission is critical for prevention of relapse. Chimeric antigen receptor T (CAR-T) cell therapy redirects genetically modified immune cells to fight against hematologic malignancies. However, given the high relapse rate after CAR-T cell therapy, MRD monitoring by traditional techniques cannot accurately quantify the disease burden, nor can they perform high-sensitivity in-depth monitoring. Further treatment options are still controversial at such a time point when the flow cytometry (FC) results show MRD < 0.01%, while tumor clones remain. Next-generation sequencing (NGS) - MRD screens out patient-specific T/B cell receptors and accurately and quantitatively monitor patient-specific tumor cells (up to 10E-6) to reveal accurate and highly sensitive MRD levels, plus provide more timely intervention criteria. Method Between June 2016 and June 2020, we retrospectively enrolled 27 patients who achieved complete remission at the first 4-week evaluation after CAR-T cell therapy. BM samples were harvested and stored before CAR-T cell infusion and 10-154 days after infusion. We evaluated 63 specimens in total 27 patients, plus tracked immunoglobulin (IG) sequencing rearrangement by next-generation sequencing (NGS) in 20 patients. Next, we classified 17 patients into high-risk (HR, NGS-MRD positive) and low-risk (LR, NGS-MRD negative) groups according to the lower limit of NGS detection (10E-6), as well as performed leukemia-free survival (LFS) and overall survival (OS) analysis. Results At least one trackable IG clonal sequence was identified in the pre-CAR-T BM specimens from 20/27 of the cases analyzed. The two diagnostic samples with low DNA quantity, in addition to five samples lacking a dominant index sequence were excluded from further analysis. We measured the MRD in 63 samples, using a threshold of 0.01%, the determination of the presence (or absence) of leukemia was concordant in 46/63 (73%) of the samples. Discordance between NGS and FC was identified in 17/63 samples (27%). Of the 20 patients for whom trackable sequences were identified, 17 with detectable clonal index sequences on +30 days were included in our subsequent analysis cohort (Figure 1). The baseline characteristics of the patients are presented in Table 1. NGS identified 9 out of the 17 patients (52.9%) whose level of MRD was > 0.01%, but MRD negative as measured by FC. This controversial group (n=9) had inferior LFS than those whose MRD was less than 0.01% by NGS (median, 56 days vs. 219 days, p = 0.037) (Figure 2A). The OS rate was comparable between the two groups (p = 0.129) (Figure 2B). Patients with positive NGS-MRD at day 30 had comparable LFS compared with those with negative NGS-MRD (p = 0.103) (Figure 3A). For subgroup analysis, we further analyzed the influence of HSCT on prognosis of HR and LR patients. Of the total 11 patients in HR group, seven non-HSCT subjects all relapsed within three months, with a median LFS of 38 days. In contrast, the remaining four HSCT patients in HR subgroup had a significantly better LFS than the non-HSCT patients (median, 495 days vs. 38 days, p = 0.003) (Figure 3B). These four patients also exhibited better OS to that of the non-HSCT group (median, 768 days vs 409 days, p = 0.006) (Figure 3C). As for the LR cohort, no significant difference was found in LFS and OS between the HSCT and non-HSCT groups (LFS: p = 0.782, OS: p = 0.782) (Median LFS and OS data not shown). All of the evidence demonstrated that NGS-MRD early after CAR-T is an efficient prognostic factor, especially for early relapse prediction. Early post-CAR-T NGS-MRD status may be recommended for HSCT timing. As such, for HR patients, whose NGS-MRD results were positive, timely HSCT may significantly improves the prognosis and therefore is highly recommended at an early point after CAR-T cell therapy. Conclusion NGS-MRD elaborately demonstrated the tumor dynamics. NGS-MRD evaluated early after CAR-T cell therapy is an efficient prognostic factor, and timely HSCT is highly recommended for NGS-MRD positive patients at an early point after CAR-T cell therapy. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.
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