Molidustat Targets a Synthetic Lethal Vulnerability in APC-Mutant Colorectal Cancer through GSTP1 and PHD2 Co-Inhibition

Reviewer #1 (Public review):

Summary:

The authors aimed to uncover novel therapeutic vulnerabilities in APC-mutant colorectal cancer (CRC), which constitutes the majority of CRC cases. They hypothesized that modulating oxygen-sensing pathways (via PHD inhibition) could disrupt adaptive stress responses in these tumours.

Strengths:

The study employs a powerful, two-pronged approach to identify Molidustat's targets. By using both Thermal Proteome Profiling (TPP) and an orthogonal chemical proteomic competition assay, the authors provide compelling evidence that GSTP1 is a genuine, direct off-target, effectively addressing the common limitation of indirect effects in proteomic screens.

Weaknesses:

(1) In Figure 1, the current data rely on a single guide RNA (sgRNA). To make the data solid, at least two independent sgRNAs targeting different regions of PHD2 should be used.

(2) Figure 3E: Asn205 site should be mutated to prove that whether Molidustat inhibits GSTP1 activity via Asn205 or not.

(3) Figure 5B and 5C: The metabolic imbalance phenotype observed upon dual knockout of PHD2 and GSTP1 requires rescue experiments to confirm on-target specificity.

Reviewer #2 (Public review):

Summary:

The authors aimed to determine Molidustat targets and the potential utility of these findings. They clearly demonstrate that Molidustat interferes with GSTP1 and some other proteins on top of PHD2. They also demonstrate that PHD2 deletion is not sufficient to recapitulate Molidustat effects in cells and proteomes. Finally, they demonstrate synthetic lethality in organoids for Molidustat and APC deletion.

Strengths:

The data on Molidustat proteomes, GSTP1 binding, inhibition and metabolic health of organoids is really clear. All biochemical, docking and omic data are really strong. The potential impact of these findings could be the use of Molidustat in APC null tumours and awareness of potential off-target effects.

Weaknesses:

A main but minor weakness is that Molidustat also inhibits other PHDs, although these are less expressed. PHD1 has been shown to control the cell cycle and be expressed in the colon, where it is needed for viability. Although this does not explain the lack of effect of other PHD inhibitors, it does warrant some discussion. The use of MTT is not very good to detect viability when it measures metabolism; this also needs to be discussed and perhaps supplemented with colony or cell number measurements.

Reviewer #3 (Public review):

In this paper, the authors revealed that Molidustat can induce a dose-dependent increase in Caspase-3/7 activity in the HT29 cell line, which is an APC-mutant colorectal cancer cell line. More importantly, they found that targeting PHD2 alone cannot cause cell death. By using thermal proteome profiling (TPP) and orthogonal chemical proteomic competition assays, they determined GTSP1 as a previously undiscovered off-target of Molidustat. They also revealed that combined PHD2 and GSTP1 loss leads to an increase in intracellular ROS and apoptosis. Moreover, they evaluated the effects of Molidustat in colonic organoids and showed that Molidustat has a high selectivity for colonic organoids with activated WNT signaling and/or KRAS pathway alterations, and this effect is not reproduced by hydroxylase inhibition alone, providing a new potential approach to targeting both PHD2 and GTSP1 for the treatment of APC-mutant CRC.

Specific comments:

(1) What is the possible molecular mechanism of dual GSTP1/PHD2 loss, inducing cell death?

(2) Can the authors mutate the binding site of Molidustat on GTSP1 to verify the in silico docking results?

(3) Evidence for Molidustat inhibiting PHD2 activity or stabilising HIF-1α should be provided.