Inhibition of DOT1L and PRMT5 promote synergistic anti-tumor activity in a human MLL leukemia model induced by CRISPR/Cas9

Only Hematopoietic Stem and Progenitor Cells from Cord Blood Are Susceptible to Malignant Transformation by MLL-AF4 Translocations

“…PCR analyses of genomic DNA revealed signals of MLL-AF4 translocations in three out of 10 and MLL-AF9 translocations in four out of eight performed experiments with different donors, demonstrating an easy translation of our previously used CRSIPR/Cas9-system to adult cells ( Figure 1 B). Sanger sequencing revealed specific fusion sequences comparable to our huCB approach ( Figure 1 C,D) [ 14 ]. These results demonstrate that we were able to induce MLL translocations with high frequency in HSPCs derived from huBM by using genome engineering.…”

“…Our models are based on patient-specific sequences and share not only genetical but also morphological, phenotypical and transcriptomic attributes of KMT2A r leukemias and hereby closely parallel the nature of the disease. Previously, we could demonstrate that by using CRISPR/Cas9 in huCB the creation of an authentic infant KMT2A r model based on endogenous oncogene activation was possible to unravel the pathogenesis of KMT2Ar leukemogenesis in vitro [ 14 ]. In this study, we successfully transferred our genetic tool to HSPCs derived from huBM and could again demonstrate the feasibility to generate both MLL-AF4 and MLL-AF9 translocations with high efficiency in an adult system.…”

“…CD34 + HSPCs were isolated (human CD34 MicroBead Kit UltraPure, Miltenyi, Bergisch Gladbach, Germany) either from fresh huCB obtained from the Department of Gynecology (IRB approval 751/2015BO2) or from huBM obtained from the Department of Hematology and Oncology of the University Hospital Tuebingen (IRB approval 309/2018BO2) and maintained in culture as previously described [ 14 ]. CRISPR/Cas9 was used to target patient-specific MLL-AF4 and - AF9 breakpoints for KMT2A r model induction and T7 endonuclease I assay (NEB, Ipswich, MA, USA) was performed to evaluate cutting efficiencies [ 14 ].…”

“…CD34 + HSPCs were isolated (human CD34 MicroBead Kit UltraPure, Miltenyi, Bergisch Gladbach, Germany) either from fresh huCB obtained from the Department of Gynecology (IRB approval 751/2015BO2) or from huBM obtained from the Department of Hematology and Oncology of the University Hospital Tuebingen (IRB approval 309/2018BO2) and maintained in culture as previously described [ 14 ]. CRISPR/Cas9 was used to target patient-specific MLL-AF4 and - AF9 breakpoints for KMT2A r model induction and T7 endonuclease I assay (NEB, Ipswich, MA, USA) was performed to evaluate cutting efficiencies [ 14 ]. Rearrangements were identified via PCR (AccuPrime Pfx DNA Polymerase, Thermo Fisher Scientific, Waltham, MA, USA), reverse transcriptase (RT)-PCR, Fluorescence in situ hybridization (FISH, Cytocell MLL ( KMT2A ) Breakapart Probe, Cambridge, UK), karyotyping and Sanger sequencing (Seqlab, Goettingen, Germany) as previously described [ 14 ].…”

PRMT5 function and targeting in cancer

“…Significantly, PRMT5 inhibition in established tumors could disable mechanisms protecting tumor cells from DNA damaging stress brought on by radiotherapy and chemotherapy. Indeed, PRMT5 depletion/inhibition sensitizes tumor cells to drugs inducing the DDR, exemplified by cytarabine (MLL-rearranged leukemia, [132]), PARP inhibitors (AML, [69]) and camptothecin, a topoisomerase inhibitor [92].…”