Introduction of STAG2 Mutation in an iPSC Model of Transient Abnormal Myelopoiesis Mimics Down Syndrome Myeloid Leukemia

Background Children with Down syndrome (DS) have a high risk for acute myeloid leukemia (DS-ML). Genomic characterization of DS-ML blasts showed the presence of unique mutations in GATA1, an essential hematopoietic transcription factor, leading to the production of a truncated from of GATA1 (GATA1s). GATA1s together with trisomy 21 is sufficient to develop a pre-leukemic condition called transient abnormal myelopoiesis (TAM). Approximately thirty percent of these cases progress into DS-ML by acquisition of additional somatic mutations in a step-wise manner. We previously developed a model for TAM by introducing disease-specific GATA1 mutation in trisomy 21 induced pluripotent stem cells (iPSCs) leading to the production of N-terminally truncated short form of GATA1 (GATA1s) (Barwe et al., 2021). In this study, we introduced co-operating mutation in STAG2, a member of the cohesin complex recurrently mutated in DS-ML but not in TAM, and evaluated its effect on hematopoietic differentiation. Methods Two different iPSC lines with trisomy 21 with or without GATA1 mutation as described in Barwe et al., 2021, were used. CRISPR/Cas9 gene editing was performed to introduce STAG2 mutation to generate a knockout of STAG2. Hematopoietic differentiation of these iPSC lines was performed using STEMdiff Differentiation kit. ProteinSimple Wes system was used for western blot analysis. Multi-dimensional flow cytometry was used for immunophenotypic analysis of megakaryoblasts cultured in lineage expansion media for 5 days. Multi-lineage colony forming potential was assessed by Methocult colony forming assay using day 10 hematopoietic stem progenitor cells (HSPCs). Results Hematopoietic differentiation of GATA1 and STAG2 double mutants in two independent trisomy 21 iPSC lines confirmed GATA1s expression and the loss of functional STAG2 protein (Fig. 1A). GATA1s expressing HSPCs collected on day 12 post differentiation showed reduced erythroid (CD71+CD235+) and increased megakaryoid (CD34+CD41+ within CD41+ compartment) and myeloid (CD18+CD45+) population compared to disomy 21 HSPCs with wild-type GATA1, consistent with our previous study (Fig. 2B). STAG2 knockout HSPCs showed higher erythroid population (P=0.033 and 0.016 in T21-1S and T21-2S respectively) and reduced myeloid population while it had no significant effect on the megakaryoid population in both iPSC lines. The GATA1s/STAG2 knockout HSPCs showed reduced erythroid, but higher megakaryoid and myeloid population compared to wild-type HSPCs. Strikingly, the immature megakaryoid population was significantly higher in the double mutant HSPCs compared to single mutant alone in both iPSC lines (P=0.005 and 0.004 for T21-1GS and T21-2GS respectively), indicating that the STAG2knockout co-operated with GATA1s for increasing megakaryoid population. The trisomy 21 iPSC line with wild-type GATA1 developed CFU-GEMM (colony-forming unit granulocyte erythroid macrophage megakaryocyte), CFU-GM (CUF granulocyte-macrophage) and BFU-E (burst-forming unit erythroid) colonies in Methocult. GATA1 mutation, unlike STAG2 mutation, inhibited the formation of CUF-GEMM and BFU-E colonies. The number of CFU-GM colonies in T21-2GS was significantly reduced compared to T21-2G (Fig. 1C, p=0.002). Lineage expansion and immunophenotyping of these HSPCs in megakaryocyte-specific media showed that these cells expressed markers closely resembling DS-ML immunophenotype. Of note, the myeloid markers, CD13 and CD11b are the only two markers expressed on majority of DS-ML blasts compared to TAM blasts (Karandikar et al., 2001) (Yumura-Yagi et al., 1992). The percentage of CD13 and CD11b expressing cells was higher in megakaryoblasts expanded from iPSC lines with STAG2 GATA1 double mutant (Fig. 1D). The number of cells expressing CD117, a stem cell marker shown recently to be involved in DS-ML progression, were highest in T21-1GS and T21-2GS lines when compared to their respective isogenic family of GATA1 mutant lines. Conclusion GATA1s and STAG2 knockout co-operated to increase the megakaryoid population and the percentage of cells expressing DS-ML markers. We have developed a model system representing DS-ML, which can be used for understanding the individual and synergistic contribution of these gene mutations in disease initiation and progression. Figure 1 Figure 1. Disclosures Barwe: Prelude Therapeutics: Research Funding. Gopalakrishnapillai: Geron: Research Funding.

Transient Abnormal Myelopoiesis with a Novel GATA1 Mutation in a Child with Down Syndrome: A Case Report and Brief Review

Transient abnormal myelopoiesis (TAM) is a unique entity seen in children with Down syndrome (DS) with 10 to 20% risk of developing myeloid leukemia in the first 5 years of life. We report a 2 months old male infant with DS detected to have hyperleukocytosis on routine preoperative workup for cyanotic congenital heart disease. Peripheral blood and bone marrow aspiration showed blasts, and next-generation sequencing detected a novel GATA1 mutation, and a diagnosis of TAM was confirmed in this child. This mutation has not been reported in TAM in the literature earlier to the best of our knowledge.

Transient Abnormal Myelopoeisis and Mosaic down Syndrome in a Phenotypically Normal Newborn

Transient abnormal myelopoiesis (TAM) is a common and potentially fatal neonatal complication of newborn babies with Down syndrome (DS). Children born with mosaic DS are also at risk of developing TAM. However, due to their variable phenotypes, early identification of patients with mosaic DS may be difficult; thus, early diagnosis of TAM is just as challenging. In this report, we describe a case of a phenotypically normal newborn who presented with concerns for neonatal leukemia. The diagnosis of mosaic DS and TAM was confirmed with abnormal GATA1 mutation testing, highlighting the importance of early GATA1 mutation testing in newborn leukemia with high suspicion for TAM.

CBFA2T3-GLIS2 Fusion Gene Cause Dismal Clinical Course in Acute Megakaryocytic Leukemia of Down Syndrome

[Introduction] Acute megakaryocytic leukemia of Down syndrome (DS-AMKL) is characterized by excellent outcome with chemotherapy in contrast to non-Down syndrome-related AMKL (non-DS-AMKL). DS-AMKL and non-DS-AMKL have distinct genetic features which may underlie their different clinical characteristics. DS-AMKL is initiated by a GATA1 mutation in the transient abnormal myelopoiesis (TAM) phase and developed with further mutations of other regulators, while non-DS-AMKL is a heterogeneous group which occasionally carry chimeric oncogenes. CBFA2T3-GLIS2 fusion gene is identified in about 30% of children with non-DS-AMKL, and reported as a strong poor prognostic factor in pediatric AMKL. However, CBFA2T3-GLIS2 has never been reported in DS-AMKL and adult AMKL patients. We performed genomic analysis of DS-AMKL including atypical case with difficult clinical course. This is the first report of DS-AMKL harboring the CBFA2T3-GLIS2 fusion gene. [Case] The patient is a 1-year-old female of DS-AMKL with no prior episode of TAM. G-banding analysis revealed the karyotype both of the leukemic cells and normal tissue sample; 47, XX, +21. Chimeric genes of AML1-MTG8, CBFB-MYH, DEK-CAN, MLL-LTG4, MLL-LTG9, MLL-ENL and abnormalities of KIT and FLT3 were not detected. The chemotherapy according to the Japanese Pediatric Leukemia / Lymphoma Study Group AML-D05 protocol, gemtuzumab ozogamicin, IDA-FLAG regimen (idarubicin, fludarabine, cytarabine, filgrastim) and clofarabine-based regimen were tried, but all of them failed to achieve complete remission (CR). She underwent umbilical cord blood transplantation and relapsed on day 35 after transplantation. Once she showed a response to azacitidine, but finally she died on day 293 after transplantation. [Materials and Methods] We performed whole transcriptome sequencing (RNAseq), SNP array analysis, mutational analysis of GATA1 in 6 DS-AMKL samples, which included this refractory sample and five DS-AMKL samples with GATA1 mutations. To analyze gene expression profiling, we applied the hierarchical clustering method and principal component analysis. [Results] RNA sequencing analysis identified a fusion gene involving exon 10 of CBFA2T3 and exon 2 of GLIS2 gene in this refractory sample. This fusion gene was a result of a cryptic inversion on chromosome 16 and the in-frame fusion of both genes. The fusion transcript was validated by reverse transcription-polymerase chain reaction (RT-PCR) followed by Sanger sequencing. Though SNP array analysis confirmed 21 trisomy, it did not identify other copy number aberrations. PCR analysis did not detect GATA1 mutation in this refractory sample, which can be identified in other DS-AMKL samples. Expression analysis elucidated DS-AMKL with CBFA2T3-GLIS2 fusion had distinct expression profile from DS-AMKL with GATA1 mutations. [Discussion] CBFA2T3-GLIS2 fusion is the most common chimeric oncogene identified in non-DS-AMKL children, but has never been detected in DS-AMKL patients. Patients with non-DS-AMKL, especially holding CBFA2T3-GLIS2 fusion gene, have poorer outcomes than DS-AMKL. DS-AMKL patients generally have GATA1 mutations, show high sensitivity to chemotherapy, and can be treated with less intensive chemotherapy. However, our case had no GATA1 mutation and could not achieve CR despite intensive chemotherapy and transplantation. Thus, it is suggested this fusion gene caused the resistance to chemotherapies including hematopoietic stem cell transplantation in our case. Therefore, our case suggests patients with DS-AMKL should be surveyed genomic investigations including RNAseq and mutational analysis of GATA1 to identify their molecular biological subtypes before treatments are initiated. In case that fusion genes are detected in DS-AMKL patients, they must undergo highly intense chemotherapies, looking ahead to transplantation from the beginning of the treatment. Moreover, in case of harboring CBFA2T3-GLIS2 fusion gene, some potential therapies have been proposed, so that efficacy of such new therapies should be validated in a cell line-derived xenograft or patient-derived xenograft model. [Conclusion] DS-AMKL is generally known to show superior outcome, but DS-AMKL without GATA1 mutation and with CBFA2T3-GLIS2 fusion gene shows resistance to chemotherapies. For DS-AMKL patients, it is desirable to perform genomic analysis including RNAseq before chemotherapy. Disclosures No relevant conflicts of interest to declare.