Endosome-microautophagy targeting chimera (eMIATAC) for targeted proteins degradation and enhance CAR-T cell anti-tumor therapy

Abstract

Rationale: Since oncogene expression products often exhibit upregulation or abnormally activated activity, developing a technique to regulate abnormal protein levels represent a viable approach for treating tumors and protein abnormality-related diseases. Methods: We first screened out eMIATAC components with high targeted degradation efficiency and explored the mechanism by which eMIATAC induced target protein degradation, and verified the degradation efficiency of the target protein by protein imprinting and flow cytometry. Next, we recombined eMIATAC with some controllable elements to verify the regulatable degradation performance of the target protein. Subsequently, we constructed eMIATAC that can express targeted degradation of AKT1 and verified its effect on GBM cell development in vitro and in vivo. Finally, we concatenated eMIATAC with CAR sequences to construct CAR-T cells with low BATF protein levels and verified the changes in their anti-tumor efficacy. Results: we developed a system based on the endosome-microautophagy-lysosome pathway for degrading endogenous proteins: endosome-MicroAutophagy TArgeting Chimera (eMIATAC), dependent on Vps4A instead of lysosomal-associated membrane protein 2A (LAMP2A) to bind to the chaperone Hsc70 and the protein of interest (POI). The complex was then transported to the lysosome by late endosomes, where degradation occurred similarly to microautophagy. The eMIATACs demonstrated accuracy, efficiency, reversibility, and controllability in degrading the target protein EGFP. Moreover, eMIATAC exhibited excellent performance in knocking down POI when targeting endogenous proteins in vivo and in vitro. Conclusions: The eMIATACs could not only directly knock down abnormal proteins for glioma treatment but also enhance the therapeutic effect of CAR-T cell therapy for tumors by knocking down T cell exhaustion-related proteins. The newly developed eMIATAC system holds promise as a novel tool for protein knockdown strategies. By enabling direct control over endogenous protein levels, eMIATAC has the potential to revolutionize treatment for cancer and genetic diseases.

Keywords: Autophagy degradation; Cancer therapy; Targeted protein degradation; eMIATAC.

Figure 1

Robustness of 2×KFERQ-vhhGFP4 for EGFP degradation. (A) Left, the mean fluorescence intensity (MFI) exhibited by mKE, KE, or CTME after intracellular expression was displayed. Right, distribution of fluorescent cells in 293T cells stably expressing mKE or KE. (B) MFI of mKE and KE in U251 and MB-231 cells. (C) Schematic representation of constructs used: KG (KFERQ-vhhGFP4), 2KV (2×KFERQ-vhhGFP4), VK (vhhGFP4), V2K (control with KFERQ mutated to KV), and CTMV (positive control). (D) Western blot analysis of HEK-293T cells co-transfected with EGFP and indicated constructs. EGFP levels were significantly reduced in cells expressing 2KV and CTMV compared to KG, VK, V2K, and untransfected control (Empty). (E) MFI of intracellular EGFP in HEK-293T cells co-transfected with EGFP and indicated constructs. Data are representative of three independent experiments (mean ± SD). (F) Co-immunoprecipitation (Co-IP) assay to detect the interaction between EGFP and constructs. Western blot analysis shows strong interaction between mKV (2×KFERQ) and EGFP, while KV showed weak interaction likely due to rapid degradation of the KV-EGFP complex in 293T cells.

Figure 2

Targeted Knockdown of EGFP by KV and Its Effect on Membrane and Nuclear Proteins. (A) Western blot analysis of HEK-293T cells co-transfected with EGFP and KV at different ratios (KV: EGFP). The results suggest an optimal knockdown effect at ratios of 1:1 or 2:1. EGFP protein levels (B) and MFI (C) at different time points within cells after mKV and KV treatment in 293T cells. (D) Western blot analysis of HEK-293T cells treated with increasing doses of His-TAT-tagged KV. A dose-dependent decrease in EGFP levels is observed. (E) Western blotting showed that the efficiency of TAT-KV reached its peak at about 4h (200μM), and EGFP returned to the baseline level at about 8h, indicating that TAT-KV may maintain a short duration of action and has a good regulatory performance. (F) Confocal microscopy images of HEK-293T-YFP-mRFP cells co-expressed with mKV or KV for 24 hours. The images show the localization of mRFP (red) and lysosomes (green; stained with Lysotracker). Pearson correlation analysis revealed limitations in using this method to quantify the substantial aggregation of YFP within lysosomes. Scale bar = 25 µm. (G) Schematic representation of constructs used: NLS-EGFP (nuclear localized EGFP; NEGFP) and CAAX-EGFP (membrane-anchored EGFP; CEGFP). Co-expression with mKV or KV in 293T cells was performed. The specific knockdown effect of KV was higher on CEGFP (52.5%) compared to NEGFP (30.8%) but lower than EGFP (64.1%).

Figure 3

Vps4A Dependence and LAMP2A Independence in eMIATAC-Mediated EGFP Degradation. (A) Left, Intracellular protein levels of mKE or KE after LAMP2A knockout. Right, Western blot analysis of LAMP2 knockout cells treated with mKV or KV. KV retained its ability to degrade EGFP compared to the control group. (B) Western blot analysis of LAMP2A knockdown cells treated with KV. Rab7A knockdown did not affect KV-mediated EGFP degradation. (C) Left, Intracellular protein levels of mKE or KE after Vps4A knockout. Middle and Right, Western blot analysis of Vps4A knockout cells treated with KV. KV no longer significantly affected EGFP protein levels. (D) MFI of 293T-mKE or 293T-KE with knockouts of intracellular Hsc70, Vps4A, or LAMP2. (E) MFI of cells with knockouts of intracellular Hsc70, Vps4A, or LAMP2. Knockouts of Hsc70 or Vps4A, but not LAMP2, significantly impaired the function of KV.

Figure 4

The eMIATAC containing regulatory elements can effectively regulate EGFP degradation. (A) Western blot analysis for differences in degradation efficiency of N- or C-terminus targeting KFERQ motif located at HSV-NS3/4A element. (B) MFI showed that E-HK and KH-E could degrade significantly with the ASV, and the KFERQ motif could achieve better degradation power at the N-terminal of HCV-NS3. (C) Schematic of eMIATAC containing HSV-NS3/4A under DMSO or ASV treatment. (D) Immunoblot analysis revealed that HK-V enabled EGFP degradation in the presence of ASV instead of DMSO. (E) KH-V and the action of ASV significantly regulated the degradation of EGFP. (F) The MFI of mKL-E, KL-E (Left), E-LK, and E-LmK (Right) under dark or blue light (470nm, 4mW/cm²) conditions. (G) Schematic of eMIATAC containing cpLOV2 under dark or blue-light treatment. (H) The regulation effect of mK/KL-V on EGFP under dark and blue light (470nm, 4mW/cm²) was determined by flow cytometry. (I) Immunoblotting analysis suggested that KL-V enabled EGFP degradation under blue-light (470 nm, 4 mW/cm²).

Figure 5

Degradation of endogenous AKT1 and pAKT by regulated eMIATAC in vivo. (A) Survival curve and tumor diameter comparison between mice treated with control eMIATAC (KA) and eMIATAC containing the inducible degradation domain (mKA). mKA treatment resulted in improved survival and smaller tumors. (B) Western blot analysis of AKT1 and pAKT levels in tumor tissues from nude mice xenografted with luciferase-infected U251 tumor cells. Samples were extracted from tumors expressing mKA and KA, respectively. (C) Flowchart for subcutaneous construction of the BRCA model and subsequent treatment in female nude mice (5 weeks old). (D) Bioluminescence imaging of nude mice xenografted with luciferase-infected MB-231 tumor cells. Mice received daily oral ASV after BRCA model construction. The left side shows mHK-A treatment and the right side shows HK-A treatment. (E) After separate flanking tumors were isolated, their volumes were measured (panel F). (G) Western blot analysis of lysed tumor tissues reveals that AKT1 and pAKT levels within HK-A expressing tumors were downregulated by ASV compared to controls.

Figure 6

The eMIATAC was used to inhibit T cell exhaustion and enhance its tumor cell killing function. (A) Overexpression of different eMIATACs into primary T cells, only KFERQ-JUN binding domain (K-S) targeting BATF can effectively reduce the protein level of BATF. (B) Effector cytokine production of primary T cells after introduction to CAR or CAR-M (n = 4). (C) The protein level of BATF in primary T cells after the introduction of CAR or CAR-M (n = 4). (D) Cytotoxic activity of CAR-T or CAR-MT against U251-luciferase at different E:T ratios (n = 3). (E) Cytotoxic activity of CAR-T or CAR-MT against U251-luciferase at different time points (n = 3). (F) The remaining proportion of U251 cells at different time points after co culturing CAR-T or CAR-MT with U251 EGFP cells at a ratio of 0.2:1 (E: T) (n = 3). (G) The remaining proportion of U251 cells after co-culturing CAR-T or CAR-MT with U251 EGFP cells at different E:T ratios (n = 3). (H) Schematic diagram of CAR-MT validation in vitro and in vivo. Intracranial bioluminescence images (I) and differences in bioluminescence intensity (J) captured on the 28th day after T cell injection in nude mice. (K) Monitoring the survival status of mice after T cell injection.