A Nonconserved Histidine Residue on KRAS Drives Paralog Selectivity of the KRASG12D Inhibitor MRTX1133.

MRTX1133 is the first noncovalent inhibitor against the KRASG12D mutant that demonstrated specificity and potency in preclinical tumor models. Here, we used isogenic cell lines expressing a single RAS allele to evaluate the selectivity of this compound. In addition to KRASG12D, MRTX1133 showed significant activity against several other KRAS mutants as well as wild-type KRAS protein. In contrast, MRTX1133 exhibited no activity against both G12D and wild-type forms of HRAS and NRAS proteins. Functional analysis revealed that the selectivity of MRTX1133 toward KRAS is associated with its binding to H95 on KRAS, a residue that is not conserved in HRAS and NRAS. Reciprocal mutation of amino acid 95 among the three RAS paralogs resulted in reciprocal change in their sensitivity toward MRTX1133. Thus, H95 is an essential selectivity handle for MRTX1133 toward KRAS. Amino acid diversity at residue 95 could facilitate the discovery of pan-KRAS inhibitors as well as HRAS and NRAS paralog-selective inhibitors. SIGNIFICANCE: The nonconserved H95 residue on KRAS is required for the selectivity of the KRASG12D inhibitor MRTX1133 and can be exploited for the development of pan-KRAS inhibitors.

The Nrf1 transcription factor is induced by patulin and protects against patulin cytotoxicity.

Patulin is a mycotoxin produced by a variety of molds that is found in various food products. The adverse health effects associated with exposure to patulin has led to many investigations into the biological basis driving the toxicity of patulin. Nevertheless, the mechanisms through which mammalian cells resists patulin-mediated toxicity is poorly understood. Here, we show that loss of the Nrf1 transcription factor renders cells sensitive to the acute cytotoxic effects of patulin. Nrf1 deficiency leads to accumulation of ubiquitinated proteins and protein aggregates in response to patulin exposure. Nrf1 expression is induced by patulin, and activation of proteasome genes by patulin is Nrf1-dependent. These findings suggest the Nrf1 transcription factor plays a crucial role in modulating cellular stress response against patulin cytotoxicity.

The deubiquitinating enzyme USP7 regulates the transcription factor Nrf1 by modulating its stability in response to toxic metal exposure.

The nuclear factor E2-related factor 1 (Nrf1) transcription factor performs a critical role in regulating cellular homeostasis as part of the cellular stress response and drives the expression of antioxidants and detoxification enzymes among many other functions. Ubiquitination plays an important role in controlling the abundance and thus nuclear accumulation of Nrf1 proteins, but the regulatory enzymes that act on Nrf1 are not fully defined. Here, we identified ubiquitin specific protease 7 (USP7), a deubiquitinating enzyme, as a novel regulator of Nrf1 activity. We found that USP7 interacts with Nrf1a and TCF11-the two long protein isoforms of Nrf1. Expression of wildtype USP7, but not its catalytically defective mutant, resulted in decreased ubiquitination of TCF11 and Nrf1a, leading to their increased stability and increased transactivation of reporter gene expression by TCF11 and Nrf1a. In contrast, knockdown or pharmacologic inhibition of USP7 dramatically increased ubiquitination of TCF11 and Nrf1a and reduction of their steady state levels. Loss of USP7 function attenuated the induction of Nrf1 protein expression in response to treatment with arsenic and other toxic metals, and inhibition of USP7 activity significantly sensitized cells to arsenic treatment. Collectively, these findings suggest that USP7 may act to modulate abundance of Nrf1 protein to induce gene expression in response to toxic metal exposure.