Optimizing establishment of high need pediatric Acute Myeloid Leukemia (AML) patient derived xenograft (PDX) models Free

Introduction: Pediatric acute myeloid leukemia (pAML) patient-derived xenograft (PDX) models are vital for pre-clinical evaluation of new agents. The use of new strains of immunodeficient mice (IDM), including NSGS, NRGS, NBSGW, MISTRG/MISTRG6, may improve the low engraftment rates of human myeloid cells. In collaboration with the Children's Oncology Group (COG), we have generated the largest known collection of serially passaging pAML PDX models in the US using these IDM strains (currently 43 pAML models, https://pdxportal.research.bcm.edu). Despite advancements, most established pAML PDX models only represent a few common molecular categories. We lack PDX models for many high-risk pAML subtypes. Here, we aim to evaluate the efficacy of utilizing newer IDM strains of mice and preconditioning with busulfan to optimize engraftment and establish additional serially passaging high-need pAML PDX models.

Methods: Patient samples from rare genomic subtypes of pAML, provided by COG, were selected for their high-need genetic fusions (CBFA2T3::GLIS2, DEK::NUP214, ETV6::LMBR1, ETV6::NIPBL, MECOM, NUP98::KDM5A, NUP98::NSD1). These genetic fusions were selected based on possible availability of targeted agents, lack of available PDX models, and/or dismal current event free survival. Patients contributing samples were enrolled on AAML0531 or AAML1031 trials. One day before sample thaw, NRGS and MISTRG mice are conditioned with intraperitoneal busulfan (30-40 mg/kg). After thaw, samples are analyzed for viability and AML flow markers using human-specific antibodies (hCD33-PE, hCD45-APC-Cy7, hCD117-BV421). Mice are then tail vein injected (TVI) with 2x10⁵ cells/mouse. Mice are monitored for clinical appearance daily. At 4-8 weeks after TVI, engraftment is tracked via flow cytometry of peripheral blood (PB) every 2-4 weeks. Upon reaching 5-10% PB engraftment or signs of clinical illness, PB, bone marrow (BM), spleen (SP), and spine (SPI) are harvested and analyzed to measure disease burden, and TVI is done with same strain/conditioning for secondary recipients. Mice that do not reach the engraftment threshold by 26 weeks following TVI have tissues harvested to assess for occult engraftment. AML cells expanded in PDX models are stored for future use and genomic validation.

Results: Twelve patient samples were suitable for use, with >50% cells viable at thaw. Of these, 4 samples achieved successful engraftment. CBFA2T3::GLIS2 and DEK::NUP214 successfully engrafted in 3^rd passage recipients, NUP98::NSD1 in the 2^nd, and NUP98::KDM5A in the 1^st. Three of the four engrafting samples had >70% viability at the time of thaw. Cells from primary xenografts of NUP98::NSD1 and DEK::NUP214 were injected into multiple strains, and time to meeting endpoint criteria differed among the mouse strains. NUP98::NSD1 average survival times were as follows: NRGS (186 days), NRGS conditioned (177 days), NSGS (175 days), NBSGW (173 days), and MISTRG (136 days). For DEK::NUP214, NSGS (212 days), NRGS conditioned (212 days), MISTRG conditioned (211 days), and NRGS (174 days). CBFA2T3::GLIS2 and NUP98::KDM5A NRGS mice showed no difference in survival between conditioned and unconditioned groups. Among engrafted mice across all strains, BM disease burden consistently exceeded that of other tissues, likely due to the infrequent nature of identifying extramedullary disease in these genomic subtypes of pAML. Engrafted cells from mice were viably banked, enabling future reestablishment of models and downstream applications. CBFA2T3::GLIS2 showed the highest cell expansion (517 fold increase), followed by DEK::NUP214 (82 fold increase) and NUP98::NSD1 (48 fold increase). All collected cells from NUP98::KDM5A were used to attempt a 2^nd transplant.

Conclusion: Four of 12 viable samples achieved successful engraftment (33%). Among these 4 samples, the commercially available NSGS strain worked well for initial engraftment and yielded the greatest cell expansion. Importantly, some samples required >200 days for engraftment to occur regardless of conditioning and strain used. Conditioning NRGS and MISTRG strains does not necessarily enhance engraftment but does add potential toxicity and regulatory burdens. Ongoing work to identify optimal engraftment factors for high-need pAML genetic subtypes will enhance PDX model generation and expedite novel agent pre-clinical testing. Viably frozen cells from established models are available free of charge through the BCM PDX portal.