STAG2 mutation preserves a stem cell phenotype in FLT3-ITD-mediated MDS to leukemia transformation

Abstract Acute myeloid leukemia with myelodysplastic related changes (AML-MRC) subtype is diagnosed when patients present leukemia with known history of hematological malignancies or chemotherapy/radiotherapy treatment. However, a panel of MDS-defining mutations, including STAG2, is highly indicative of AML-MRC subtype, even when de novo AML mutation like NPM1c is detected. Among all the AML subtypes, AML-MRC often arises in older patients and harbors a poor prognosis with a 5-year overall survival rate of <30%. STAG2 mutations are found in 14-20% of AML-MRC cases and are suggested to reside within a dominant clone during the pre-leukemia phase, MDS to leukemic transformation, and persist during remission. STAG2, a cohesin complex member, maintains the integrity of the 3D genome partitioning structure known as topologically structural domains. Loss of Stag2 impairs the access and engagement of key hematopoietic transcription factors such as PU.1 to their target genes. FLT3 is mutated through an internal tandem duplication (ITD) in 20-25% of AML, driving aberrant STAT5/AKT signaling and leukemogenesis. Analysis of paired samples from patients that transformed from MDS to AML showed acquisition of Ras signaling pathway mutations (~30%) including FLT3ITD, during the transformation. So far, murine modelling of ITD showed marked myeloid progenitor expansion and hematopoietic stem cell (HSC) exhaustion in various co-mutant settings. However, there is yet a model to mimic the genetic evolution of AML-MRC with STAG2 as the founding mutation. Here we present a sequential mutagenesis murine model of co-Stag2/Flt3ITD mutation, using pIpC-inducible Mx1Cre and tamoxifen-inducible Flpo recombinase. To induce the mutation and mimic MDS and then leukemia, we deleted Stag2 via Mx1Cre and waited for 4 months (MDS phase), before activation of ITD mutation via Flpo, and start the transformation phase. After activating both mutations, mice were followed for another 4 months before analyzing the hematopoietic stem and progenitor compartment (HSPC). In FLT3ITD,we observed depletion of HSC and expansion of myeloid biased progenitors (MPP3 & GMP) as previously reported. In contrast, sequential Stag21stITD2nd mutation preserves the HSC population and has reduced expansion of myeloid progenitors. Through scRNAseq analysis, we confirmed transcriptionally defined HSC population in the Stag21stITD2nd sample, but not in the FLT3ITD. Unexpectedly, the scRNAseq analysis showed that Stag21stITD2nd HSPC has myeloid differentiation capacity, which is blocked in FLT3ITD. We then performed bulk RNAseq on purified GMP cells at 2 weeks post ITD activation and found that Stag21stITD2nd GMP cells failed to activate the JAK-STAT pathway. We are currently performing detailed genomic analysis and functional validation to determine the differentiation capacity change driven by Stag2 mutation. On the other hand, we further characterized the mutant HSCs detected in the Stag21stITD2nd sample. Using flow cytometry, we found that the Stag21stITD2nd has decreased proportion of EPCR+ HSCs. Upon transplantation, the Stag21stITD2nd HSC failed to engraft in lethally irradiated recipients, which indicates a loss of normal stem cell function. To determine how Stag2 mutation preserves the HSC population, we performed Brdu analysis on Stag2 mutant or wild-type HSCs at the end of the MDS phase. We found that the Stag2 mutation leads to an increased HSC cycling before acquiring the Flt3ITD mutation, suggesting increased stem cell proliferation. Finally, we performed competitive transplantation of the MDS phase BM and then initiated the transformation in the recipients. At 16 weeks post-transplant, we found that the FLT3ITD cells reconstitute mostly the myeloid progenitor compartment while the Stag21stITD2nd BM reconstitute across the HSPC, including the HSC. This confirms that the cell-autonomous mechanism is associated with the cellular composition change detected in Stag21stITD2nd vs Flt3ITD mutant. Overall, our data highlights how pre-existing Stag2 mutation alters the cellular composition and behavior post transformation. This model will not only shed light on the AML-MRC pathogenesis but also create a pre-clinical testing platform with potential therapeutic relevance.