A Genomics Driven, Inducible Pluripotent Stem Cell Model of Infant Lymphoblastic Leukemia

Acute lymphoblastic leukemia in infants (iALL) is a high-risk subtype of childhood leukemia with poor survival outcomes despite intensive therapies. Rearrangement of KMT2A (KMT2A-r) occurs in 70% of cases and is associated with chemotherapy resistance, early relapse, and rapid leukemic progression, all of which contribute to a survival rate of about 40%. The most common KMT2A-r in iALL is KMT2A::AFF1 (MLL::AF4), derived from t(4;11)(q21;23). This KMT2A-r generates a driver fusion oncogene which leads to epigenetic dysregulation of target gene transcription. Infant ALL expressing KMT2A::AFF1 is also a unique cancer in that other somatic mutations are uncommon and there are no known genetic risk factors. This leukemia's cell of origin is proposed to be a very early hematopoietic precursor, with transcriptomic studies of iALL blasts showing similarities to hematopoietic stem and progenitor cells (HSPCs), multipotent progenitors and early lymphoid progenitors (ELPs). Despite this knowledge, the factors leading to leukemogenesis remain poorly understood, with research into this devastating disease hampered by the lack of a representative model of iALL Attempts at creation of an iALL models have led to a variety of results including myeloid neoplasms, lymphomas, mixed phenotypic hyperplasia or leukemia, mature B cell neoplasias. Because of these failures, much remains unknown regarding how KMT2A::AFF1 transforms early hematopoiesis and how it alters the severity of the disease.

To further understand the developmental state of the cell of origin and leukemogenesis of iALL, we have engineered a highly controlled human inducible pluripotent stem (iPS) cell model of KMT2A-r iALL. Using an established doxycycline (dox) regulated platform, we have generated multiple iALL driver transgene constructs, including an epitope tagged human KMT2A::AFF1 cassette and a human-mouse KMT2A::Aff1 cassette. This system targets the dox-regulated transgene and dox regulatory machinery to the endogenous AAVS1 locus into non-diseased human iPS cell lines using CRISPR mediated targeting via homology directed repair. The non-diseases human iPS cells allow for mimicking of the silent mutational landscape found in patient samples. Correct targeting of the KMT2A::Aff1 transgene was confirmed via real-time reverse transcriptase-polymerase chain reaction (RT-PCR). These cells tightly express the KMT2A::Aff1 transgene only in response to dox treatment in both undifferentiated iPS cells and after differentiation to hematopoietic cells.

Through directed differentiation we have also produced functional HSPCs from iPS cells with multilineage differentiation capacity. Flow cytometry analysis demonstrated enrichment of CD45+CD34+ cells by day 10 of differentiation. Further flow cytometry analysis of colony forming unit assays revealed functional enrichment in lympho-myeloid (CD45RA+) differentiated cells, along with formation of monocyte (CD15+) and erythroid (CD235+) colonies. We also used the 10x Chromium system to perform single-cell RNA sequencing of the differentiated cells. The scRNAseq transcriptomic data was analyzed using Seurat in R. Transcriptomic data confirmed the cells' differentiation ability as they clustered into distinct populations representing erythrocytes, megakaryocytes, and monocytes.

In conclusion, this model has allowed us to recapitulate hematopoietic ontogeny with the ability to control iALL induction at specific developmental stages. We are utilizing single cell genomics to investigate transcriptomic changes during hematopoietic differentiation and after expression of the KMT2A oncogenic driver. We will compare these results to published data sets of single cell RNA sequencing data from healthy fetal HSPCs and to our existing iALL and pediatric KMT2A-r ALL single cell sequencing data sets. Our iPS cell based iALL model system provides the opportunity to investigate a range of critical and outstanding questions of iALL disease initiation, progression, and treatment.

Farooqi:Pacific Biosciences: Honoraria, Other: travel support; Bayer: Honoraria; 10X Genomics: Honoraria, Other: travel support. Guest:Amgen: Current Employment; Jazz Pharmaceuticals: Speakers Bureau; Syndax Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees.