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Extramedullary infiltration (EMI) is a concomitant manifestation that may indicate poor outcome of acute myeloid leukemia (AML). The underlying mechanism remains poorly understood and therapeutic options are limited. Here, we employed single-cell RNA sequencing on bone marrow (BM) and EMI samples from a patient with AML presenting pervasive leukemia cutis. A complement C1Q+ macrophage-like leukemia subset, which was enriched within cutis and existed in BM before EMI manifestations, was identified and further verified in multiple patients with AML. Genomic and transcriptional profiling disclosed mutation and gene expression signatures of patients with EMI that expressed high levels of C1Q. RNA sequencing and quantitative proteomic analysis revealed expression dynamics of C1Q from primary to relapse. Univariate and multivariate analysis demonstrated adverse prognosis significance of C1Q expression. Mechanistically, C1Q expression, which was modulated by transcription factor MAF BZIP transcription factor B, endowed leukemia cells with tissue infiltration ability, which could establish prominent cutaneous or gastrointestinal EMI nodules in patient-derived xenograft and cell line–derived xenograft models. Fibroblasts attracted migration of the C1Q+ leukemia cells through C1Q–globular C1Q receptor recognition and subsequent stimulation of transforming growth factor β1. This cell-to-cell communication also contributed to survival of C1Q+ leukemia cells under chemotherapy stress. Thus, C1Q served as a marker for AML with adverse prognosis, orchestrating cancer infiltration pathways through communicating with fibroblasts and represents a compelling therapeutic target for EMI.

The pyruvate kinase (PK) is a rate-limiting glycolytic enzyme catalyzing the dephosphorylation of phosphoenolpyruvate to pyruvate. M2 form of PK (PKM2) is expressed during embryogenesis and is the predominant form in tumors of different types. In contrast to the essential role of PKM2 in solid tumors, much less is known about the effects of PKM2 in hematopoietic cells and the development of leukemia. Here we found that PKM2 is modified by small ubiquitin-like modifier 1(SUMO1), which can be reduced by a SUMO1-specific protease SENP1 in hematopoietic cells. SUMOylation induced nuclear localization and conformation change from tetramer to dimer of PKM2. Importantly, SUMOylation of PKM2 is prevalent in a variety of leukemic cell lines as well as primary samples from patients with hematologic malignancies. In consistency, predominant nuclear localization and dimeric forms of PKM2 in leukemic cells were observed. Using in vitro SUMOylation reaction-coupled liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS), we identified K270 lysine residue of PKM2 as the SUMOylation target. Replacement of endogenous PKM2 with mutant PKM2K270 showed a significant shift of PKM2 from tetramer to dimer. To investigate the potential leukemogenic effect of PKM2 SUMOylation, murine hematopoietic progenitor 32D clone 3 (32Dcl3) transfectants expressing wild type(WT) or mutant PKM2K270 were generated and G-CSF-induced differentiation was evaluated by morphology appearance and expression of myeloid associated surface markers CD11b and Gr-1. The results showed that expression of WT PKM2 but not mutant PKM2K270 significantly blocked myeloid differentiation. Further investigations revealed that SUMO1 modification of PKM2 at K270 is essential in mediating the interaction between PKM2 and Runt-related transcription factor 1(RUNX1), a master transcriptional factor implicated in the differentiation of hematopoietic cells. This interaction led to a downregulation of RUNX1 during G-CSF-induced myeloid differentiation of 32D cells, which could be abrogated by expression of mutant PKM2K270. Collectively, these data indicated that SUMOylated PKM2 blocks myeloid differentiation through suppressing RUNX1. These findings reveal a novel nonmetabolic function of PKM2 in modulating myeloid differentiation and highlight the critical role of SUMOylation in leukemogenesis. Disclosures No relevant conflicts of interest to declare.

Abstract 1433 BACKGROUND High levels of 2-HG in serum were found among AML cases with IDH1/IDH2 mutations. However, the impact of 2-HG levels on biological characteristics and clinical outcomes has not been systematically evaluated in a large cohort of AML patients. METHODS Serum 2-HG levels in 699 patients with hematologic malignancies, including 405 AML patients, were measured by GC-TOFMS. Clinical prognostic power of 2-HG and genetic risk factors associated with 2-HG were also evaluated in AML patients. RESULTS Among all patients with hematopoietic malignancies investigated, high serum 2-HG levels were observed only in AML group. 64 out of 405 (15.8%) AML patients displayed aberrantly higher levels (7.14±2.11μg/ml) of 2-HG. Compared to cases with normal 2-HG (3.65±1.02μg/ml), these patients showed a higher prevalence in AML-M0/M1 subtypes, a closer correlation with mutated IDH1/2 genes, a distinct gene expression profile and an aberrant DNA methylation status in bone marrow blasts. Additionally, the patients with high 2-HG were associated with higher serum levels of α-ketoglutarate (α-KG) and glutamate, suggesting presence of impaired metabolic pathway involved in the biosynthesis of 2-HG. Univariate and multivariate analyses indicate that high level of 2-HG is among the most significant negative indicators for complete remission (CR) rate, overall survival (OS) and event-free survival (EFS) either in all AML cases or in cases with cytogenetically normal AML (CN-AML). CONCLUSIONS Serum 2-HG is an independent prognostic marker in AML. Patients with high 2-HG had significantly higher frequency of IDH1/IDH2 gene mutations and unfavorable prognosis compared to those with normal 2-HG. Disclosures: No relevant conflicts of interest to declare.