ALBERT

Description: Simultaneous measurement of cell lineage and cell fates is a longstanding goal in biomedicine. Here we describe EMBLEM, a strategy to track cell lineage using endogenous mitochondrial DNA variants in ATAC-seq data. We show that somatic mutations in mitochondrial DNA can reconstruct cell lineage relationships at single cell resolution with high sensitivity and specificity. Using EMBLEM, we define the genetic and epigenomic clonal evolution of hematopoietic stem cells and their progenies in patients with acute myeloid leukemia. EMBLEM extends lineage tracing to any eukaryotic organism without genetic engineering.

Description: Introduction: Loss-of-function mutations in Runt-related transcription factor 1 (RUNX1) are commonly found in both germline and somatic hematopoietic malignancies and confer particularly poor prognosis in AML. However, it remains unclear how RUNX1 functions during hematopoietic and leukemic development, particularly because RUNX1 mutations alone are not sufficient to cause myeloid malignancy and some models show that RUNX1 mutations confer hematopoietic stem cell defects. Recently, mouse models have shown that RUNX1-deficient neutrophils upregulate NFκB activity, and hematopoietic stem and progenitor cells (HSPCs) with overactive inflammatory pathways gain competitive advantage under chronic inflammation. Thus, we hypothesized that while RUNX1 mutations impair normal HSPC function, inflammation may select for or rescue RUNX1 mutant HSPCs. Methods: To interrogate the effect of RUNX1 loss in human CD34+ HSPCs, we disrupted the RUNX1 locus using CRISPR/Cas9 and AAV6-mediated homology directed repair. Importantly, by using an AAV6 vector that carries arms of homology flanking a fluorescent reporter expression cassette, we are able to track and isolate cells edited at the RUNX1 locus for in vitro and in vivo functional analyses and for molecular characterization using RNA-seq and ATAC-seq. Results: First, we used this system to evaluate the functional consequences of RUNX1 knockout (KO) in human CD34+ HSPCs. Loss of RUNX1 caused early erythroid-megakaryocytic differentiation arrest and skewing toward monocytic differentiation. RUNX1 KO cells demonstrated decreased proliferation, cell cycle arrest, and reduction in serial replating potential in vitro. In competitive transplantation experiments in NSG mice, RUNX1 KO engraftment decreased over time in both primary and secondary transplant, revealing a competitive disadvantage. Second, ATAC-seq peak motif analysis showed that PU.1 and NFκB motifs are more accessible upon RUNX1 KO whereas GATA, TAL1, and RUNX motifs were less accessible. Similarly, gene set enrichment analysis of transcriptional data confirmed the broad upregulation of NFκB-mediated inflammatory programs; downregulation of GATA1-dependent heme metabolism and platelet development pathways; and downregulation of MYC- and E2F-dependent cell cycle programs. These observations imply that RUNX1 directs cell fate decisions by recruiting and activating lineage-specific hematopoietic transcription factors and augmenting stem cell proliferation programs. We next sought to determine which cytokines are sufficient to drive RUNX1 KO cell expansion. RUNX1 KO cells not only expanded preferentially in NSG mice expressing human SCF, GM-CSF, and IL-3 (NSGS mice), but also were no longer defective in competitive transplants in these mice. Further, treatment with IL-3 was sufficient to significantly expand RUNX1 KO cells in vitro. Flow cytometry revealed that the IL-3 receptor CD123 is upregulated in RUNX1 KO cells compared to control. Similarly, RUNX1-mutant AML patient samples express higher levels of CD123 than RUNX1-wildtype AML patient samples. Finally, evaluation of publicly available RUNX1 ChIP-seq of bone marrow CD34+ HSPCs revealed that RUNX1 directly binds the promoter of CD123. Ongoing efforts are aimed at determining whether targeting CD123 and IL-3 signaling may be a viable therapeutic approach for the prevention or treatment of RUNX1-mutant myeloid malignancies. Conclusion: In summary, we established a RUNX1-deficient human HSPC model not only to evaluate the role of RUNX1 in hematopoiesis, but also to characterize intrinsic and extrinsic factors involved in RUNX1-deficient clonal expansion and leukemic transformation. We show that RUNX1 KO causes monocytic skew at the expense of erythro-megakaryocytic potential and severely limits HSC engraftment and expansion in vivo. Molecular profiling reveals that these effects are associated with dysregulation of both transcription factor activity and cytokine signaling. However, exposure to IL-3 rescues RUNX1-deficient cell proliferative defects in vitro and competitive engraftment defects in vivo. This hypersensitivity to IL-3 signaling is mediated in part by increased expression of the IL-3 receptor CD123. These findings reveal how RUNX1 mutations may initially behave in a deleterious manner but can ultimately confer an advantage to HSPCs under certain environmental conditions. Disclosures Majeti: CD47 Inc.: Divested equity in a private or publicly-traded company in the past 24 months; Gilead Sciences: Divested equity in a private or publicly-traded company in the past 24 months, Patents & Royalties; Kodikaz Therapeutic Solutions Inc: Membership on an entity's Board of Directors or advisory committees.

Description: Introduction: Acute myeloid leukemia (AML) is associated with a poor prognosis even with aggressive treatments including high dose chemotherapy. While most patients enter clinical remission, these remissions are often short-lived leading to chemotherapy-resistant relapsed disease that accounts for the majority of deaths. We undertook a meta-analysis of published datasets consisting of 142 genotyped paired diagnosis-relapse AML samples to understand the genetic evolution of AML between the two disease states. This analysis determined that a plurality of cases exhibited the same mutations at diagnosis and relapse, and that genetically stable clones were associated with an increased probability of relapse. The finding that many cases exhibited no clonal genetic evolution upon relapse, yet exhibited chemotherapy resistance, lead us to hypothesize that epigenetic evolution plays a significant role in AML relapse. Here, our objective was to investigate the epigenetic evolution and cis and trans regulatory elements that correlate with AML relapse. Methods: We identified 27 paired diagnosis and relapse specimens from patients treated at Stanford with high dose chemotherapy regimens. Leukemic blasts, and in some cases leukemia stem cell (LSC)-enriched fractions, were purified by FACS. Cells were then analyzed through a multi-omic platform including genotyping with a myeloid malignancy targeted panel, RNA-seq, and ATAC-seq to obtain a molecular and chromatin accessibility profile of each sample. The resulting data set was analyzed to investigate epigenetic evolution in relapsed AML. Results: Genotyping analysis of banked AML specimens identified a similar pattern of genetic evolution as our meta-analysis, with several samples exhibiting the same mutations at diagnosis and relapse. We used an epigenetic matrix of chromatin accessibility data obtained from purified cell populations within the hematopoietic hierarchy and implemented this with the CIBERSORT algorithm to map the regulatory programs active in diagnosis and relapsed AML blasts. This analysis revealed a general trend of epigenetic states associated with more primitive cells (such as hematopoietic stem and progenitor cells) active at relapse, as opposed to more differentiated myeloid cell programs active at diagnosis. Focusing further on samples with no genetic changes between the two disease states, we observed several samples with substantial epigenetic evolution at relapse, with AML blasts shifting from a more differentiated myeloid cell profile to that of stem and progenitor cells. These changes were associated with a loss of accessibility in PU.1 and CEBPα transcription factor motifs, with a corresponding increase in GATA and RUNX motifs, suggesting epigenetic remodeling contributes to relapse even in the absence of genetic changes. We have additionally identified various categories of relapse samples in our cohort that share similar epigenetic profiles relating to genotype; NPM1 and FLT3 double mutant samples, for example, shared active chromatin accessibility features. Given the key importance of LSCs in AML pathogenesis and their potential role in chemotherapy resistance, we further undertook an analysis of cellular subpopulations enriched for these cells in a subset of our sample cohort. ATAC-seq analysis of CD34+CD38- cell fractions revealed these cells share many epigenetic features between samples, yet also have distinct regulatory programs from those active in leukemia non-stem cells and exhibit similar epigenetic reprogramming between diagnosis and relapse. This analysis further indicates that epigenetic evolution at relapse occurs at the single cell level, rather than reflecting selection of cellular subpopulations at relapse. Ongoing work involves identifying the specific regulatory programs upregulated in relapse samples, and LSCs specifically, to understand how these programs contribute to relapse at the gene regulatory level. Conclusion: Our results indicate a substantial role for epigenetic evolution in AML, with the activation of more primitive stem and progenitor programs upon relapse. We have also identified epigenetic classifications for several relapse samples that correspond to genotype and characterized the regulatory programs associated with relapse. We hope this work will permit a deeper understanding of the evolutionary factors that guide AML relapse. Disclosures Majeti: Zenshine Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees; Coherus BioSciences: Membership on an entity's Board of Directors or advisory committees; CircBio Inc.: Research Funding; BeyondSpring Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees; Stanford University: Patents & Royalties: pending patent application on CD93 CAR ; Forty-Seven Inc.: Divested equity in a private or publicly-traded company in the past 24 months; Kodikaz Therapeutic Solutions Inc.: Membership on an entity's Board of Directors or advisory committees; Gilead Sciences, Inc.: Patents & Royalties: inventor on patents related to CD47 cancer immunotherapy.