Abstract
High-throughput sequencing describes multiple alterations in individual tumors, but their functional relevance is often unclear. Clinic-close, individualized molecular model systems are required for functional validation and to identify therapeutic targets of high significance for each patient. Here, we establish a Cre-ERT2-loxP (causes recombination, estrogen receptor mutant T2, locus of X-over P1) based inducible RNAi- (ribonucleic acid interference) mediated gene silencing system in patient-derived xenograft (PDX) models of acute leukemias in vivo. Mimicking anti-cancer therapy in patients, gene inhibition is initiated in mice harboring orthotopic tumors. In fluorochrome guided, competitive in vivo trials, silencing of the apoptosis regulator MCL1 (myeloid cell leukemia sequence 1) correlates to pharmacological MCL1 inhibition in patients´ tumors, demonstrating the ability of the method to detect therapeutic vulnerabilities. The technique identifies a major tumor-maintaining potency of the MLL-AF4 (mixed lineage leukemia, ALL1-fused gene from chromosome 4) fusion, restricted to samples carrying the translocation. DUX4 (double homeobox 4) plays an essential role in patients’ leukemias carrying the recently described DUX4-IGH (immunoglobulin heavy chain) translocation, while the downstream mediator DDIT4L (DNA-damage-inducible transcript 4 like) is identified as therapeutic vulnerability. By individualizing functional genomics in established tumors in vivo, our technique decisively complements the value chain of precision oncology. Being broadly applicable to tumors of all kinds, it will considerably reinforce personalizing anti-cancer treatment in the future.
Highlights
High-throughput sequencing describes multiple alterations in individual tumors, but their functional relevance is often unclear
In proof of principle studies, we demonstrated that MCL1 silencing in acute lymphoblastic leukemia (ALL) and acute myeloid leukemia (AML) patient-derived xenograft (PDX) models correlates to response to pharmacological MCL1 inhibition
Transduction efficiencies were typically well below 30% (Table S2), putatively indicating a single viral integration per genome according to literature[24], leading to homogenous expression levels of Cre-ERT2 (Fig. S1a), minimal toxicity and neglectable leakiness in all samples, overcoming one of the challenges of TRE-based inducible expression systems[16]
Summary
High-throughput sequencing describes multiple alterations in individual tumors, but their functional relevance is often unclear. Clinic-close, individualized molecular model systems are required for functional validation and to identify therapeutic targets of high significance for each patient. We establish a Cre-ERT2-loxP (causes recombination, estrogen receptor mutant T2, locus of X-over P1) based inducible RNAi- (ribonucleic acid interference) mediated gene silencing system in patient-derived xenograft (PDX) models of acute leukemias in vivo. Functional studies have largely been restricted to cancer cell lines, which often fall short in predicting the role of alterations in individual human tumors[7]. We confirmed a tumor-maintaining potency of the MLL-AF4 fusion protein in PDX models in vivo and used the technique to identify DDIT4L as therapeutic targets in PDX ALL carrying the recently described DUX4-IGH translocation
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