Inducible transgene expression in PDX models in vivo identifies KLF4 as a therapeutic target for B-ALL

Abstract

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.

A reporter system for enriching CRISPR/Cas9 knockout cells in technically challenging settings like patient models

“…Having established the surrogate reporter, we next tested our system in two PDX ALL models, ALL-199 and ALL-256 (patient characteristics are detailed in Supplemental Table S3 as published previously 21 – 23 ). Compared to leukemia cell lines, PDX ALL cells are substantially more challenging in handling as they are hard to transduce and reluctant to grow in vitro 21 , 22 .…”

“…Procedures were performed following manufacturer’s instructions; in brief, each capillary was loaded with protein lysate and electrophoresis was performed; capillaries were processed to attach all proteins to the capillary wall and incubated with a single antibody; results were measured as emission curves from each capillary and “Western-Blot-like presentations” calculated thereof using the Compass software (ProteinSimple), including quantification. Final “Western-Blot-like presentations” appear clearly different from conventional Western Blots as described 23 ; due to very high sensitivity, equal loading is hard to achieve, but also not required, as protein amounts are calculated with high sensitivity and reliability. Primary antibody against LYN was purchased from R&D systems (AF3206, Minneapolis, MN, USA), FLAG from R&D systems (MAB8529, Minneapolis, MN, USA) and β-ACTIN from Novus biologicals (NB600-501SS, Littleton, CO, USA).…”

“…Cloning of the the pCDH-based lentiviral sgRNA expression vector, with mTagBFP or mCherry expressed under the control of the EF1α promoter was previously described 23 . The sgRNA sequences (see supplementary Table S1 , left panel) were synthesized (Eurofins MWG-Operon, Ebersberg, Germany), annealed and ligated into the vector using BbsI.…”

“…For Cas9 expression, a DNA fragment containing the cDNA of spleen focus forming virus (SFFV) promoter (Addgene plasmid #27348), humanized Staphyococcus pyogenes Cas9 (hSpCas9 PX330, Addgene plasmid #42230) and a T2A-linked mTaqBFP fluorochrome (pTagBFP-C vector, PF171, Envrogen, Lawrenceville, NJ) or a truncated form of the human nerve growth factor receptor (hNGFR) was amplified by PCR and cloned into the pCDH vector (Addgene Plasmid #72263) as previously described 23 .…”

“…PDX ALL-265 and ALL-199 were established and the NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ (NSG) mouse model performed as described 21 – 23 ; Lentiviruses were produced and cells infected as described 21 – 23 ; in PDX and NALM-6 cells, when creating the stable LYN KO cell lines, transduction efficiency was at or below 40% to aim for single integrations of lentiviruses into the host genome 17 . After lentiviral transduction, PDX cells were injected intravenously into NSG mice for amplification 24 h post infection, or maintained in vitro 4 days and enriched for transgene expression before injection.…”