Lentiviral expressed biodegrader affects the LMO2 multi-protein complex in T cell lines

The T cell lines Jurkat (LMO2-), KOPT-K1 (LMO2+), and P12-Ichikawa (LMO2+) cells were infected with lentivirus packaging plasmids and transfer vector (TLCV2-VH576-L10-CRBN) for 16 hours followed by 2 μg/ml doxycycline induction for 9, 24, and 48 hours. Western blotting analysis was used to detect LMO2, endogenous CRBN, and VH576-L10-CRBN levels after lentivirus infection. α-tubulin was used as an internal loading control for Western blotting analysis (panel A). The level of LMO2 and proteins associated with the LMO2 transcription complex (TAL1, E47, Lyl-1, GATA3, and LDB1) was determined after KOPT-K1 cells were infected with TLCV2-VH576-L10-CRBN followed by 2 μg/ml doxycycline induction for 9, 24, and 48 hours (panel B). Cyclophilin-b were used as an internal loading control.

LMO2 Abd degraders and effects on cellular protein

Structure-activity observations of LMO2-binding compounds derived using iDAb VH576 in competitive, cell-based BRET (designated Abd compounds) contributed to a general structure of the LMO2-binding compounds (panel A). Based on broad tolerance to chemical modification at position R’, two PROTACs were synthesised, one bearing the VHL ligand (Abd-VHL, panel B) and the other bearing thalidomide (the CRBN ligand) (Abd-CRBN, panel C). Each PROTAC thus comprises the LMO2-binding ligand, a linker and an E3 ligase ligand.

KOPT-K1 and Jurkat cells were treated with different concentration of Abd-degraders at 0, 5, 10, and 20 μM for 24 hours, protein extracts prepared and fractioned by SDS-PAGE followed by Western blotting with the antibodies indicated by each panels D and E. Abd-CRBN was used on cells in panels D and Abd-VHL in panels E. Cell treated with 1% DMSO was used as a control and β-actin was used as an internal loading control for the Western blotting analysis. The viability of KOPT-K1 cells were treated with 15 μM Abd-CRBN (panel F) or 15 μM Abd-VHL (panel G) for 0, 2, 4, 6, and 24 hours viability determined using CellTiter-Glo assay. Data are presented as a relative to luminescence at 0 hours, normalized to 100%. Determination of DC50 values of Abd-CRBN and Abd-VHL after 24 hours treatment in KOPT-K1 was determined using CellTiter-Glo calculated by GraphPad Prism 9.0 software (panel H and I, respectively). All the values were presented as the average values relative to cell viability values in control (DMSO treated cells) normalized to 100%. Data represent mean + SEM (n=3). Luciferase cell-based report assays were used to determine the degradation of the proteins after transfected HEK293T cells with different Renilla luciferase (Rluc8) reporter plasmids (pEF-LMO2-Rluc8, pEF-MAX-Rluc8, pEF-RHOBWT-FL, pEF-KRSWT-Fl-RLuc8, pEF-GFP2-Rluc8, pEF-ISL1-Rluc8, and pEF-Zn268) followed by Abd-CRBN (panel J) and Abd-VHL (panel K) treatment at 0, 10 and 20 μM for 24 hours. The potential degradation of pEF-Tal-Rluc8 (panel L) and pEF-LMO2-Rluc8 (panel M) was determined by luciferase cell-based report assays after the treatment with Abd-CRBN or Abd-VHL art 0, 10 and 20 μM for 24 hours. Data represent mean + SEM (n=3).

Proteins associated within the LMO2 transcription complex are co-degraded with the LMO2 PROTACs

LMO2 expressing (KOPT-K1 and CCRF-CEM) and non-expressing T cells (Jurkat and DND-41) were treated with Abd-degraders and protein extracts were prepared for Western blot, detecting LMO2, TAL1, E47, Lyl-1, GATA3, LDB1 proteins (cyclophilin-b was used as an internal loading control) after cells were treated with different concentration of Abd- degraders at 0, 5, 10, 15, and 20 μM for 24 hrs. Panel A: Western blotting analysis with KOPT-K1 and CCRF-CEM extracts and panel B with Jurkat and DND-41 extracts. Panel C: KOPT-K1 and CCRF-CEM were treated with Abd-degraders at 20 μM for 2, 4, 6, and 24 hours. Western blotting analysis data show LMO2, TAL1 and E47 proteins expression in KOPT-K1 and CCRF-CEM that was affected by treatment. Panel D: The involvement of proteasome machinery in protein complex degradation was investigated in KOPT-K1 and CCRF-CEM with either proteosome inhibitors or by competing the potential of the Abd degraders with free E3 ligase ligand. Western blot data show LMO2, TAL1, and E47 expression in KOPT-K1 and CCRF-CEM after the treatment with or without inhibitors followed by Abd-compounds. Inhibitors used were the proteasome inhibitor epoxomicin (0.8 μM), or CRBN inhibitors (10 μM thalidomide) or free VHL ligand. Cyclophilin-b was used as an internal loading control for Western blotting analysis.

Loss of LMO2 protein inhibits T-ALL cell growth

LMO2 expressing T cells were treated with 20 μM Abd-CRBN or Abd-VHL for 24 and 48 hours and protein extracts were prepared for Western blot to detect LMO2 protein, RAS protein as negative control and β-actin was used as an internal loading control. Western blotting analysis data show LMO2 expression in KOPT-K1 (panel A), PF-382 (panel B), P12-Ichikawa (panel C), CCRF-CEM (panel D), MOLT-16 (panel E), and LOUCY (panel F) that was affected by treatment with the LMO2 PROTAC compounds. Comparative cell numbers were determined from increase in viability determined using the CellTiter-Glo assay. Data represent mean + SEM (n=3). LMO2 expressing cell lines tested were KOPT-K1 (G, t(11;14)(p13;q11)), PF-382 (H), P12-Ichikawa (I, t(11;14)(p13;q11)), CCRF-CEM (J), MOLT-16 (K), and LOUCY (L). LMO2 non-expressing T cells were also treated with 20 μM Abd-CRBN or Abd-VHL for 24 and 48 hours after which relative luminescence was determined using the CellTiter-Glo assay. Cells tested were Jurkat (panel M), DND-41 (panel N), ALL-SIL (panel O), SUPT-1 (panel P), and RPMI8402 (panel Q). Data represent mean + SEM (n=3).

Proteomic profiling of T-ALL cells after treatment with Abd PROTAC degrader

LMO2 expressing T cells (KOPT-K1) and LMO2 non-expressing T cells (Jurkat and RPMI8402) were treated in duplicate with 15 μM Abd-VHL for 24 hours. Panel A: Floating bar plots of LMO2 protein levels measured by targeted proteomics in control (DMSO treated) (grey bars) and Abd-VHL treated (red bars) in KOPT-K1, Jurkat and RPMI8402 cells. Panel B: Western blotting analysis of LMO2 levels in KOPT-K1 cells when treated with DMSO only and Abd-VHL for 24 hours. β-actin protein detection was used as an internal loading control for Western blotting analysis. Panel C: Heatmap displaying enriched terms from GSEA in KOPT-K1, Jurkat, and RPMI8402 cells after Abd-VHL treatment compared to control treatment (DMSO), based on global proteomics analysis. The enrichment score ranges from -2.5 (blue) to 2.5 (yellow). Panel D and E: Volcano plot of proteomic changes following Abd-VHL treatment compared to control treatment (DMSO) in KOPT-K1 (D) and Jurkat (E). The plots are based on the fold change (log2FC) and p-value (-log10(p-value)). Respectively, the yellow or grey circles indicate proteins with statistically significant or non-significant up-regulation/down-regulation. The cut-off for the volcano plots is log2FC=0.585, p-value=0.05). Panel F: a tabulation of statistically significant proteins in KOPT-KI, represented as yellow circles in Panel D compared to Jurkat cells. The data are presented as fold change (log2FC) and p-value (-log10(p-value)).

Caspase and PARP cleavage in cells after treatment with Abd degraders indicative of apoptosis initiation

KOPT-K1 and Jurkat cells were treated with Abd-CRBN or Abd-VHL and effects on viability and programmed cell death were analysed at different times. Panel A: Time course of progressive expression of caspases after the treatment with compounds was assayed for caspase 3/7 levels using Caspase-Glo® 3/7. Cells were treated with 20 μM Abd-CRBN or Abd-VHL followed by cell culture up to 30 hours. Cells treated with 1% DMSO in culture medium was used as control. Data represent mean + SEM (n=3). KOPT-K1 and Jurkat cells were treated with 20 μM Abd-CRBN (panel B) or Abd-VHL (panel C) and cultured for 24 or 48 hours. Cell extracts were made and proteins subjected to Western blotting analysis with specific antibodies for detection of LMO2, caspase 3, cleaved caspase 3, caspase 7, cleaved caspase 7, PARP, and cleaved PARP. β-actin protein detection was used as an internal loading control for Western blotting analysis.