FGFR1 Fusion Genes Combine with RUNX1 Mutations to Drive Leukemogenesis

8p11 myeloproliferative syndrome is a myeloid tumor characterized by FGFR1 fusion gene. Its progression is fast, prognosis is bad and pathogenesis is not clear. We screened 10 cases of FGFR1 rearrangement (1 new fusion gene TFG-FGFR1), and 8 of them performed second generations of target exon capture and resequencing. The results showed that RUNX1 gene mutation occurred in 6 cases, with a mutation rate of 75%. The pretest results showed that FGFR1 fusion gene could significantly activate the downstream signaling pathway of FGFR1, and then promote cell proliferation, while RUNX1 gene mutation could block cell differentiation. It is presumed that FGFR1 gene fusion and RUNX1 mutation could lead to leukemia formation. Based on our previous work, we will explore the synergistic pathogenicity, the differences of variable opposite genes and possible mechanisms of FGFR1 fusion gene and RUNX1 mutation in vitro based on cell biology and animal models in vivo, and explore the therapeutic effect of targeted drugs in such diseases. This topic will help clarify the synergistic pathogenic effect of FGFR1 fusion gene and RUNX1 mutation and its related mechanisms, and provide theoretical and experimental evidence for the diagnosis and treatment of this disease. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

RUNX1 Expression Can be Complementary to RUNX1 Mutation in MDS Prognostication

RUNX1 is a member of the core-binding factor family of transcription factors and is imperative for establishing definitive haematopoiesis. Mutated RUNX1 is an adverse risk factor for myelodysplastic syndrome (MDS) and acute myeloid leukaemia (AML). Meanwhile, high expression of RUNX1 is correlated with dismal prognosis in cytogenetically normal AML patients and critical for maintaining leukemic stem cells across AML genetic subgroups. However, the clinical relevancy of RUNX1 expression in MDS patients remains elusive. This study aimed to investigate the prognostic and biologic impacts of RUNX1 expression in MDS patients. We recruited 341 primary MDS patients who had enough bone marrow (BM) samples for RNA and next-generation sequencing. We first examined the difference in RUNX1 expression among patients with wild RUNX1 and N-terminal or C-terminal RUNX1 mutation. Among the 341 patients, 54 (15.8%) harboured RUNX1 mutation, 15 (27.8%) in N-terminal and 39 (72.2%), C-terminal. Patients with C-terminal RUNX1 mutant had higher RUNX1 expression than those with N-terminal RUNX1 mutant or wild RUNX1 (p<0.001 ). Patients were then divided into two groups with higher- and lower-RUNX1 expression (median as cut-off). Higher RUNX1 expression was closely associated with lower platelet counts, higher blast percentages in the BM and peripheral blood and complex karyotypes at diagnosis. Patients with higher-RUNX1 expression were more frequently categorized into higher-risk groups based on the revised international prognosis scoring system (IPSS-R). Higher RUNX1 expression was intimately associated with ASXL1, NPM1, RUNX1, SRSF2, STAG2, TET2, TP53, and ZRSR2 mutations, whereas lower RUNX1 was associated with SF3B1 mutation. Regarding survival, we first examined the impact of RUNX1 mutation on survival in this cohort. As expected, patients with mutated RUNX1 had significantly inferior leukaemia-free survival (LFS) and overall survival (OS) than those with unmutated RUNX1 (p=0.007, and p=0.008, respectively). We then explored the effects of RUNX1 expression on patients' survival. Patients with higher RUNX1 expression had significantly inferior LFS and OS than those with lower expression (both p<0.001, Figure 1a and 1b). We further interrogated RUNX1 expression and mutation statuses for risk stratification. The higher-RUNX1 group consistently had shorter LFS and OS than the lower-RUNX1 group no matter RUNX1 was mutated or not (Figure 1c-1f). Subgroups analysis revealed the same findings in IPSS-R lower-risk (very low, low, and intermediate-risk) and IPSS-R higher-risk (high and very high risk) subgroups (all p<0.05). Moreover, time-dependent ROC curves indicated that RUNX1 expression had better predictive power for LFS and OS than RUNX1 mutation. In multivariate analysis, higher RUNX1 expression appeared as an independent adverse risk factor for LFS and OS irrespective of age, IPSS-R, and mutations in ASXL1, EZH2, RUNX1, SF3B1, SRSF2, STAG2, TET2, and TP53 (Table). The prognostic significance of RUNX1 expression was further validated in two external public cohorts, GSE 114922 and GSE15061. Patients with higher-RUNX1 expression consistently had significantly inferior survival than those with lower-RUNX1 expression (Figure 2). Bioinformatic analysis revealed that higher-RUNX1 patients had more robust IL-17 and MAPK signallings but exhausted antioxidant activities and antimicrobial humoral responses. In summary, we present the characteristics and prognosis of MDS patients with various RUNX1 expressions and propose that RUNX1 expression can complement RUNX1 mutation in MDS prognostication, wherein patients with wild RUNX1 but high expression may need more proactive treatment. Figure 1 Figure 1. Disclosures Tien: AbbVie: Honoraria; Celgene: Honoraria, Research Funding; Novartis: Honoraria. Chou: Abbvie: Honoraria, Other: Advisory Board, Research Funding; Celgene: Honoraria, Other: Advisory Board, Research Funding; IQVIA: Honoraria, Other: Advisory Board; Pfizer: Honoraria, Other: Advisory Board; Novartis: Honoraria, Other: Advisory Board; Bristol Myers Squibb: Honoraria, Research Funding; Kirin: Honoraria, Research Funding.

Effective therapy of AML with RUNX1 mutation by co-treatment with inhibitors of protein translation and BCL2

Majority of RUNX1 mutations in AML are missense or deletion-truncation and behave as loss-of-function mutations. Following standard therapy, AML patients expressing mtRUNX1 exhibit inferior clinical outcome than those without mutant RUNX1. Studies presented here demonstrate that as compared to AML cells lacking mtRUNX1, their isogenic counterparts harboring mtRUNX1 display impaired ribosomal biogenesis and differentiation, as well as exhibit reduced levels of wild-type RUNX1, PU.1 and c-Myc. Compared to AML cells with only wild-type RUNX1, AML cells expressing mtRUNX1 were also more sensitive to the protein translation inhibitor homoharringtonine (omacetaxine) and BCL2 inhibitor venetoclax. HHT treatment repressed enhancers and their BRD4 occupancy, as well as was associated with reduced levels of c-Myc, c-Myb, MCL1 and Bcl-xL. Consistent with this, co-treatment with omacetaxine and venetoclax or BET inhibitor induced synergistic in vitro lethality in AML expressing mtRUNX1. Compared to each agent alone, co-treatment with omacetaxine and venetoclax or BET inhibitor also displayed improved in vivo anti-AML efficacy, associated with improved survival of immune depleted mice engrafted with AML cells harboring mtRUNX1. These findings highlight superior efficacy of omacetaxine-based combination therapies for AML harboring mtRUNX1.

The Plasmacytoid Dendritic Cell CD123+ Compartment in Acute Leukemia with or without RUNX1 Mutation: High Inter-Patient Variability Disclosed by Immunophenotypic Unsupervised Analysis and Clustering

Plasmacytoid dendritic cells (PDC) constitute a small subset of normal bone marrow (BM) cells but have also been shown to be present, sometimes in large numbers, in several hematological malignancies such as acute myeloid leukemia with RUNX1 mutation, chronic myelomonocytic leukemia or, obviously, blastic plasmacytoid dendritic cell neoplasms. These cells have been reported to display somewhat variable immunophenotypic features in different conditions. However, little is known of their plasticity within individual patients. Using an unsupervised clustering tool (FlowSOM) to re-visit flow cytometry results of seven previously analyzed cases of hematological malignancies (6 acute myeloid leukemia and one chronic myelomonocytic leukemia) with a PDC contingent, we report here on the unexpectedly high variability of PDC subsets. Although five of the studied patients harbored a RUNX1 mutation, no consistent feature of PDCs could be disclosed as associated with this variant. Moreover, the one normal single-node small subset of PDC detected in the merged file of six normal BM could be retrieved in the remission BM samples of three successfully treated patients. This study highlights the capacity of unsupervised flow cytometry analysis to delineate cell subsets not detectable with classical supervised tools.

Novel Germline RUNX1 Mutation Associated with Familial Thrombocytopenia as well as B-Acute Lymphoblastic Leukemia: A Case Report and Review of the Literature

Germline RUNX1 mutations lead to a rare form of autosomal-dominant familial thrombocytopenia with a predisposition for myeloid malignancies and are classified as distinct entities by the WHO. We report a case of B lymphoblastic leukemia developing in a patient with a familial RUNX1 mutation, which is a first in the literature. An FLT3-ITD mutation as well as a balanced chromosomal translocation t(1;7) was present at the time of diagnosis of leukemia, favoring the theory that additional hits or mutations are necessary for malignant transformation in patients with a germline RUNX1 mutation. The transformed disease runs an aggressive course compared to the same malignancy associated with a somatic RUNX1 mutation. Additionally, family members should be screened for the mutation, followed up clinically if they carry the mutation, and should not be used as stem cell donors to treat the affected relatives.

Different mutant RUNX1 oncoproteins program alternate haematopoietic differentiation trajectories

Mutations of the haematopoietic master regulator RUNX1 are associated with acute myeloid leukaemia, familial platelet disorder and other haematological malignancies whose phenotypes and prognoses depend upon the class of the RUNX1 mutation. The biochemical behaviour of these oncoproteins and their ability to cause unique diseases has been well studied, but the genomic basis of their differential action is unknown. To address this question we compared integrated phenotypic, transcriptomic, and genomic data from cells expressing four types of RUNX1 oncoproteins in an inducible fashion during blood development from embryonic stem cells. We show that each class of mutant RUNX1 deregulates endogenous RUNX1 function by a different mechanism, leading to specific alterations in developmentally controlled transcription factor binding and chromatin programming. The result is distinct perturbations in the trajectories of gene regulatory network changes underlying blood cell development which are consistent with the nature of the final disease phenotype. The development of novel treatments for RUNX1-driven diseases will therefore require individual consideration.