RESUMO
Systematic testing of existing drugs and their combinations is an attractive strategy to exploit approved drugs for repurposing and identifying the best actionable treatment options. To expedite the search among many possible drug combinations, we designed a combinatorial CRISPR-Cas9 screen to inhibit druggable targets. Coblockade of the N-methyl-d-aspartate receptor (NMDAR) with targets of first-line kinase inhibitors reduced hepatocellular carcinoma (HCC) cell growth. Clinically, HCC patients with low NMDAR1 expression showed better survival. The clinically approved NMDAR antagonist ifenprodil synergized with sorafenib to induce the unfolded protein response, trigger cell-cycle arrest, downregulate genes associated with WNT signaling and stemness, and reduce self-renewal ability of HCC cells. In multiple HCC patient-derived organoids and human tumor xenograft models, the drug combination, but neither single drug alone, markedly reduced tumor-initiating cancer cell frequency. Because ifenprodil has an established safety history for its use as a vasodilator in humans, our findings support the repurposing of this drug as an adjunct for HCC treatment to improve clinical outcome and reduce tumor recurrence. These results also validate an approach for readily discovering actionable combinations for cancer therapy. SIGNIFICANCE: Combinatorial CRISPR-Cas9 screening identifies actionable targets for HCC therapy, uncovering the potential of combining the clinically approved drugs ifenprodil and sorafenib as a new effective treatment regimen.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Biomarcadores Tumorais/metabolismo , Sistemas CRISPR-Cas , Carcinoma Hepatocelular/tratamento farmacológico , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Neoplasias Hepáticas/tratamento farmacológico , Animais , Apoptose , Biomarcadores Tumorais/genética , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/patologia , Proliferação de Células , Humanos , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos NOD , Camundongos Nus , Camundongos SCID , Piperidinas/administração & dosagem , Sorafenibe/administração & dosagem , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
We present a CRISPR-based multi-gene knockout screening system and toolkits for extensible assembly of barcoded high-order combinatorial guide RNA libraries en masse. We apply this system for systematically identifying not only pairwise but also three-way synergistic therapeutic target combinations and successfully validate double- and triple-combination regimens for suppression of cancer cell growth and protection against Parkinson's disease-associated toxicity. This system overcomes the practical challenges of experimenting on a large number of high-order genetic and drug combinations and can be applied to uncover the rare synergistic interactions between druggable targets.