CRISPR/Cas9 represents a valuable tool to determine protein function, but technical hurdles limit its use in challenging settings such as cells unable to grow in vitro like primary leukemia cells and xenografts derived thereof (PDX). To enrich CRISPR/Cas9-edited cells, we improved a dual-reporter system and cloned the genomic target sequences of the gene of interest (GOI) upstream of an out-of-frame fluorochrome which was expressed only upon successful gene editing. To reduce rounds of in vivo passaging required for PDX leukemia growth, targets of 17 GOI were cloned in a row, flanked by an improved linker, and PDX cells were lentivirally transduced for stable expression. The reporter enriched scarce, successfully gene-edited PDX cells as high as 80%. Using the reporter, we show that KO of the SRC-family kinase LYN increased the response of PDX cells of B precursor cell ALL towards Vincristine, even upon heterozygous KO, indicating haploinsufficiency. In summary, our reporter system enables enriching KO cells in technically challenging settings and extends the use of gene editing to highly patient-related model systems.

Chemotherapy resistance is the main impediment in the treatment of acute myeloid leukaemia (AML). Despite rapid advances, the various mechanisms inducing resistance development remain to be defined in detail. Here we report that loss-of-function mutations (LOF) in the histone methyltransferase EZH2 have the potential to confer resistance against the chemotherapeutic agent cytarabine. We identify seven distinct EZH2 mutations leading to loss of H3K27 trimethylation via multiple mechanisms. Analysis of matched diagnosis and relapse samples reveal a heterogenous regulation of EZH2 and a loss of EZH2 in 50% of patients. We confirm that loss of EZH2 induces resistance against cytarabine in the cell lines HEK293T and K562 as well as in a patient-derived xenograft model. Proteomics and transcriptomics analysis reveal that resistance is conferred by upregulation of multiple direct and indirect EZH2 target genes that are involved in apoptosis evasion, augmentation of proliferation and alteration of transmembrane transporter function. Our data indicate that loss of EZH2 results in upregulation of its target genes, providing the cell with a selective growth advantage, which mediates chemotherapy resistance.

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.

Background Clinically relevant methods are not available that prioritize and validate potential therapeutic targets for individual tumors, from the vast amount of tumor descriptive expression data. Methods We established inducible transgene expression in clinically relevant patient-derived xenograft (PDX) models in vivo to fill this gap. Results With this technique at hand, we analyzed the role of the transcription factor Krüppel-like factor 4 (KLF4) in B-cell acute lymphoblastic leukemia (B-ALL) PDX models at different disease stages. In competitive preclinical in vivo trials, we found that re-expression of wild type KLF4 reduced the leukemia load in PDX models of B-ALL, with the strongest effects being observed after conventional chemotherapy in minimal residual disease (MRD). A nonfunctional KLF4 mutant had no effect on this model. The re-expression of KLF4 sensitized tumor cells in the PDX model towards systemic chemotherapy in vivo. It is of major translational relevance that azacitidine upregulated KLF4 levels in the PDX model and a KLF4 knockout reduced azacitidine-induced cell death, suggesting that azacitidine can regulate KLF4 re-expression. These results support the application of azacitidine in patients with B-ALL as a therapeutic option to regulate KLF4. Conclusion Genetic engineering of PDX models allows the examination of the function of dysregulated genes like KLF4 in a highly clinically relevant translational context, and it also enables the selection of therapeutic targets in individual tumors and links their functions to clinically available drugs, which will facilitate personalized treatment in the future.

Abstract Objectives: Personalized therapies target individual, tumor-specific alterations identified by descriptive genomics and transcriptomics. Selecting individual targets with high therapeutic potential remains a challenging task for Molecular Tumor Boards in today's clinical routine. Functional data, which help ranking alterations for their usefulness as therapeutic targets, are scarce, especially in individual tumors and in vivo. To bridge this gap, we invented a functional genomics in vivo approach which enables prioritizing alterations with high potential as therapeutic targets. Methods: Primary tumor cells from patients with acute leukemias (AL) were grown on immune compromised mice and patient derived xenografts (PDX) genetically modified using lentiviruses. For the first time, an inducible system was established in PDX-AL models, where knockdown was induced in vivo upon feeding mice with tamoxifen. In vivo assays were performed in a competitive way, with control and gene-of-interest cells in the same animal and monitored by recombinant fluorochromes. Results: MCL-1 is an anti-apoptotic protein frequently upregulated in tumors and inhibitors against MCL-1 are tested in clinical studies. We aimed at identifying AL patients who might profit from therapy targeting MCL-1. PDX cells were transplanted and grown in mice until tumors were established before MCL-1 knockdown was induced by feeding mice with tamoxifen. Established PDX AL revealed different intensities of growth disadvantages between individual samples, ranging from weak to strong phenotypes. In general, PDX models from patients with acute myeloid leukemia (AML) were more responsive than those from patients with acute lymphoblastic leukemia (ALL). MCL-1 played an essential role in vivo in several AML cells from patients with different cytogenetics and risk factors. In sensitive PDX samples, response to MCL-1 treatment was independent from disease stage as induction of MCL-1 knockdown severely reduced AML PDX fitness at all disease stages, from minimal to advanced disease. Inhibition of MCL-1 sensitized resistant AML cells towards different drugs. All in all, we show for the first time that PDX AML cells in vivo depend on MCL-1 and that MCL-1 represents an interesting therapeutic target for some, but not all AL samples. Conclusions: Taken together, we established a technique to identify and molecularly validate genes with an essential function in individual tumors in vivo. Our technique allows prioritizing alterations for their usefulness as therapeutic targets. Our approach will streamline clinical trials in personalized medicine in the future. Citation Format: Michela Carlet Polleux, Kerstin Völse, Jenny Vergalli, Marc Schmidt-Supprian, Irmela Jeremias. A novel in vivo technique to molecularly validate potential targets for personalized therapy [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr LB-305.