Degradation of LMO2 in T cell leukaemia results in collateral breakdown of transcription complex partners and causes LMO2-dependent apoptosis

Reviewer #1 (Public review):

Summary:

The authors describe the degradation of an intrinsically disordered transcription factor (LMO2) via PROTACs (VHL and CRBN) in T-ALL cells. Given the challenges of drugging transcription factors, I find the work solid and a significant scientific contribution to the field.

Strengths:

(1) Validation of LMO2 degradation by starting with biodegraders, then progressing to chemical degrades.

(2) interrogation of the biology and downstream pathways upon LMO2 degradation (collateral degradation and apoptotic markers).

(3) Cell line models that are dependent/overexpression of LMO2 vs LMO2 null cell lines.

(4) CRBN and VHL-derived PROTACs were synthesized and evaluated.

Weaknesses:

(1) The conventional method used to characterize PROTACs in the literature is to calculate the DC50 and Dmax of the degraders, I did not find this information in the manuscript.

(2) The proteomics data is not very convincing, and it is not clear why LMO2 does not show in the volcano plot (were higher concentrations of the PROTAC tested? and why only VHL was tested and not CRBN-based PROTAC?).

(3) The correlation between degradation potency and cell growth is not well-established (compare Figure 4C: P12-Ichikawa blots show great degradation at 24 and 48 hrs, but it is unclear if the cell growth in this cell line is any better than in PF-382 or MOLT-16) - Can the authors comment on the correlation between degradation and cell growth?

(4) The PROTACs are not very potent (double-digit micromolar range?) - can the authors elaborate on any challenges in the optimization of the degradation potency?

(5) The authors mentioned trying six iDAb-E3 ligase proteins; I would recommend listing the E3 ligases tried and commenting on the results in the main text.

Reviewer #2 (Public review):

Summary:

Sereesongsaeng et al. aimed to develop degraders for LMO2, an intrinsically disordered transcription factor activated by chromosomal translocation in T-ALL. The authors first focused on developing biodegraders, which are fusions of an anti-LMO2 intracellular domain antibody (iDAb) with cereblon. Following demonstrations of degradation and collateral degradation of associated proteins with biodegraders, the authors proceeded to develop PROTACs using antibody paratopes (Abd) that recruit VHL (Abd-VHL) or cereblon (Abd-CRBN). The authors show dose-dependent degradation of LMO2 in LMO2+ T-ALL cell lines, as well as concomitant dose-dependent degradation of associated bHLH proteins in the DNA-binding complex. LMO2 degradation via Abd-VHL was also determined to inhibit proliferation and induce apoptosis in LMO2+ T-ALL cell lines.

Strengths:

The topic of degrader development for intrinsically disordered proteins is of high interest, and the authors aimed to tackle a difficult drug target. The authors evaluated methods, including the development of biodegraders, as well as PROTACs that recruit two different E3 ligases. The study includes important chemical control experiments, as well as proteomic profiling to evaluate selectivity.

Weaknesses:

The overall degradation is relatively weak, and the mechanism of potential collateral degradation is not thoroughly evaluated. In addition, experiments comparing the authors' prior work with their anti-LMO2 iDAb or Abl-L are lacking, which would improve our understanding of the potential advantages of a degrader strategy for LMO2.