An engineered chimeric toxin that cleaves activated mutant and wild-type RAS inhibits tumor growth

Abstract

<jats:p>Despite nearly four decades of effort, broad inhibition of oncogenic RAS using small-molecule approaches has proven to be a major challenge. Here we describe the development of a pan-RAS biologic inhibitor composed of the RAS-RAP1–specific endopeptidase fused to the protein delivery machinery of diphtheria toxin. We show that this engineered chimeric toxin irreversibly cleaves and inactivates intracellular RAS at low picomolar concentrations terminating downstream signaling in receptor-bearing cells. Furthermore, we demonstrate in vivo target engagement and reduction of tumor burden in three mouse xenograft models driven by either wild-type or mutant <jats:italic>RAS</jats:italic>. Intracellular delivery of a potent anti-RAS biologic through a receptor-mediated mechanism represents a promising approach to developing RAS therapeutics against a broad array of cancers.</jats:p>

RAS specific protease Induces Irreversible Growth Arrest via p27 in several KRAS Mutant Colorectal Cancer cell lines

“…categorized several colon cancer cell lines for KRAS-dependency based on survival outcomes following KRAS RNAi silencing (36). Published data from our group has shown that the KRAS Indep cell line HCT-116 was highly susceptible to RRSP growth inhibition (27). To extend this finding, the effect of RRSP processing of RAS in KRAS Dep (SW620 and GP5d ) and KRAS Indep (HCT-116, SW1463, HT-29) colon cancer cell lines was tested for relative susceptibility to RRSP ( Fig.…”

“…Recombinant RRSP-DTB and RRSP*-DTB were expressed in E. coli BL21(DE3) and purified over a HisTrap FF nickel affinity column as previously described (27). Eluted fractions were loaded onto a gravity column containing Strep-Tactin Superflow high capacity resin, followed by SUMOtag removal and size exclusion purification over a Superdex 75 column using ÄKTA protein purifier purification system as previously described (27). For intoxication, colon cancer cell lines were seeded in 6-well plates (~70% confluency) overnight, after which medium was replaced with fresh medium containing either RRSP-DTB or RRSP*-DTB and incubated at indicated timepoints at 37°C in the presence of 5% CO2.…”

“…As a result, RAS and RAP1 are unable to undergo GTP-GDP exchange or bind to their downstream effectors (33,34). Recently, RRSP engineered for in vivo delivery was shown to significantly reduce breast and colon tumor growth in xenograft mouse models (27). The potential applicability of RRSP to a broad range of cancers was also tested using the standardized National Cancer Institute (NCI) cancer cell panel (35).…”

“…The potential applicability of RRSP to a broad range of cancers was also tested using the standardized National Cancer Institute (NCI) cancer cell panel (35). Fourteen of 60 cell lines were classified as highly susceptible, 38/60 as susceptible, and only 8/60 showed low or no susceptibility to RRSP (27). The relative susceptibility of the cancer cells to RRSP did not correlate with any specific RAS mutation.…”

Untitled

“…Sci. USA, we described the development of a biologic chimeric toxin comprising RRSP fused to the translocation and receptor-binding domains of diphtheria toxin (DT B ) [7]. We showed that DT B specifically and efficiently delivers RRSP to cancer cell lines bearing the diphtheria toxin receptor HB-EGF and then abrogates cell viability and proliferation at concentrations as low as one picomolar.…”