Mutations in isocitrate dehydrogenase 1 (IDH1mut) are reported in 70-90% of low-grade gliomas and secondary glioblastomas. IDH1mut catalyzes the reduction of α-ketoglutarate (α-KG) to 2-hydroxyglutarate (2-HG), an oncometabolite which drives tumorigenesis. Inhibition of IDH1mut is therefore an emerging therapeutic approach, and inhibitors such as AG-120 and AG-881 have shown promising results in phase 1 and 2 clinical studies. However, detection of response to these therapies prior to changes in tumor growth can be challenging. The goal of this study was to identify non-invasive clinically translatable metabolic imaging biomarkers of IDH1mut inhibition that can serve to assess response. Methods: IDH1mut inhibition was confirmed using an enzyme assay and 1 H- and 13 C- magnetic resonance spectroscopy (MRS) were used to investigate the metabolic effects of AG-120 and AG-881 on two genetically engineered IDH1mut-expressing cell lines, NHAIDH1mut and U87IDH1mut. Results: 1 H-MRS indicated a significant decrease in steady-state 2-HG following treatment, as expected. This was accompanied by a significant 1 H-MRS-detectable increase in glutamate. However, other metabolites previously linked to 2-HG were not altered. 13 C-MRS also showed that the steady-state changes in glutamate were associated with a modulation in the flux of glutamine to both glutamate and 2-HG. Finally, hyperpolarized 13 C-MRS was used to show that the flux of α-KG to both glutamate and 2-HG was modulated by treatment. Conclusion: In this study, we identified potential 1 H- and 13 C-MRS-detectable biomarkers of response to IDH1mut inhibition in gliomas. Although further studies are needed to evaluate the utility of these biomarkers in vivo , we expect that in addition to a 1 H-MRS-detectable drop in 2-HG, a 1 H-MRS-detectable increase in glutamate, as well as a hyperpolarized 13 C-MRS-detectable change in [1- 13 C] α-KG flux, could serve as metabolic imaging biomarkers of response to treatment.

Purpose Targeting MET in cancer is hampered by lack of diagnostics that accurately reflect high MET signaling and dependence. We hypothesized that assays reflecting MET signaling associated protein complexes could redefine tumors dependent on MET and could add additional precision beyond genomic assessments. Experimental Design We utilized biochemical approaches, cellular viability studies and proximity ligation assays to assess MET dependence. We examined MET signaling complexes in lung cancer patient specimens (N=406) and patient-derived xenograft models of solid tumors (N=308). We evaluated response to crizotinib in a MET-amplified cohort of patient-derived xenografts models of lung cancer (N=6) and provide a case report of a lung cancer patient harboring a Δexon14 MET splice variant. Results We found the interaction of MET with the adaptor protein GRB2 is necessary for oncogenic survival signaling by MET. MET:GRB2 complexes were identified only within MET-amplified patient-derived xenograft (PDX) models and patient specimens but exhibit substantial variability. Lack of MET:GRB2 complexes was associated with lack of response to MET TKI in cell lines and PDX models. Presence of MET:GRB2 complexes can further sub type tumors with Δexon14 MET splice variants. Presence of these complexes correlated with response to crizotinib in one patient with Δexon14 MET lacking MET gene amplification. Conclusions Proximity assays measuring MET:GRB2 signaling complexes provide novel insights into MET-mediated signaling and could complement current clinical genomics-based assay platforms.

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SUMMARYChimeric antigen receptors (CARs) are modular proteins capable of redirecting immune cells toward a wide variety of disease-associated antigens. Here, we explore the effects of CAR protein sequence and structure on CAR-T cell function. Based on the empirical observation that CD20 CARs with similar sequences exhibit divergent tonic-signaling and anti-tumor activities, we devised engineering strategies that aimed to improve CAR-T cell function by tuning the intensity of tonic signaling. We found that CARs designed to exhibit low but non-zero levels of tonic signaling show robust effector function upon antigen stimulation while avoiding premature functional exhaustion by CAR-T cells. Through alterations of the CAR’s ligand-binding domain and overall protein conformation, we generated CD20 CAR variants that outperform the CD19 CAR in mouse models of human lymphoma. We further demonstrate that rational modification of protein confirmation can be generalized to improve GD2 CAR-T cell efficacy against neuroblastoma. These findings point to tonic signaling and basal T-cell activation as informative parameters to guide the rational design of next-generation CARs for cancer therapy.

These data support the development of stem cell therapies for age-associated degeneration in FFP in humans.