Dual blockade of the lipid kinase PIP4Ks and mitotic pathways leads to cancer-selective lethality

Abstract

Achieving robust cancer-specific lethality is the ultimate clinical goal. Here, we identify a compound with dual-inhibitory properties, named a131, that selectively kills cancer cells, while protecting normal cells. Through an unbiased CETSA screen, we identify the PIP4K lipid kinases as the target of a131. Ablation of the PIP4Ks generates a phenocopy of the pharmacological effects of PIP4K inhibition by a131. Notably, PIP4Ks inhibition by a131 causes reversible growth arrest in normal cells by transcriptionally upregulating PIK3IP1, a suppressor of the PI3K/Akt/mTOR pathway. Strikingly, Ras activation overrides a131-induced PIK3IP1 upregulation and activates the PI3K/Akt/mTOR pathway. Consequently, Ras-transformed cells override a131-induced growth arrest and enter mitosis where a131’s ability to de-cluster supernumerary centrosomes in cancer cells eliminates Ras-activated cells through mitotic catastrophe. Our discovery of drugs with a dual-inhibitory mechanism provides a unique pharmacological strategy against cancer and evidence of cross-activation between the Ras/Raf/MEK/ERK and PI3K/AKT/mTOR pathways via a Ras˧PIK3IP1˧PI3K signaling network.

Interrogating the protein interactomes of RAS isoforms identifies PIP5K1A as a KRAS-specific vulnerability

“…It is also worth pointing out that EFR3A , one of the most commonly negatively enriched genes identified in the CRISPR-Cas9 interactome screen, serves as a plasma membrane anchor for PI4PKIIIα, the lipid kinase that produces PtdIns(4) P , a substrate of PIP5K1A 42 . Furthermore, oncogenic RAS was recently shown to suppress the anti-proliferative effects resulting from knocking down or pharmacologically inhibiting PIP4K lipid kinases 56 .…”

“…Loss of PIP5K1A also reduced the phosphorylation of ERK induced by ectopic and endogenous oncogenic KRAS, but not NRAS or HRAS. It is possible that this is a product of cross-talk between the PI3K/AKT and MAPK pathways, for example, through wild-type RAS proteins 32 , suppressing the inhibitory PI3K1P1 by the MAPK pathway 56 , and so on, although admittedly this is a complex relationship 57 . Alternatively, the reduction of P-ERK may point to an effect on KRAS itself.…”

Thermal proteome profiling for interrogating protein interactions

“…More commonly, TPP experiments involve chemical [e.g., drug (Azimi et al, 2018;Becher et al, 2016;Hu et al, 2019;Huber et al, 2015;Kitagawa et al, 2017;Mateus et al, 2018Mateus et al, , 2016Reinhard et al, 2015;Savitski et al, 2014Savitski et al, , 2018 . Some of the perturbations can be applied in a dose-dependent manner (Becher et al, 2016) or time-dependent manner Dai et al, 2018) to improve data analysis or facilitate mechanistic understanding of the perturbation (Fig 2).…”

“…Some of the perturbations can be applied in a dose‐dependent manner (Becher et al , ) or time‐dependent manner (Becher et al , ; Dai et al , ) to improve data analysis or facilitate mechanistic understanding of the perturbation (Fig ). Using this approach, it has been possible to deconvolute drug targets (Savitski et al , , ; Huber et al , ; Reinhard et al , ; Becher et al , ; Mateus et al , , ; Kitagawa et al , ; Azimi et al , ; Hu et al , ) and enzyme substrates (preprint: Saei et al , ), study metabolic shifts (Becher et al , ; Dai et al , ; Mateus et al , ), or identify protein–protein interactions (Tan et al , ).…”

An isothermal shift assay for proteome scale drug-target identification

“…TPP has a distinct advantage over other methods in that it can be used to identify targets in lysates and intact, biologically active cells 26 . Whereas lysate based studies are more likely to reveal direct binding targets due to disruption of signaling complexes and dilution of metabolites and second messengers 27 , TPP in biologically active cells can reveal downstream events initiated by the primary target binding event, such as post-translational modifications or protein-protein interactions stimulated by target binding, and thus provide important mechanistic insights into signaling mechanisms 13,[28][29][30][31] .…”