Purpose: A cryptic inv(16)(p13.3q24.3) encoding the CBFA2T3-GLIS2 fusion is associated with poor outcome in infants with acute megakaryocytic leukemia. We aimed to broaden our understanding of the pathogenesis of this fusion through transcriptome profiling. Experimental Design: Available RNA from children and young adults with de novo acute myeloid leukemia (AML; N ¼ 1,049) underwent transcriptome sequencing (mRNA and miRNA). Transcriptome profiles for those with the CBFA2T3-GLIS2 fusion (N ¼ 24) and without (N ¼ 1,025) were contrasted to define fusion-specific miRNAs, genes, and pathways. Clinical annotations defined distinct fusion-associated disease characteristics and outcomes. Results: The CBFA2T3-GLIS2 fusion was restricted to infants <3 years old (P < 0.001), and the presence of this fusion was highly associated with adverse outcome (P < 0.001) across all morphologic classifications. Further, there was a striking paucity of recurrent cooperating mutations, and transduction of cord blood stem cells with this fusion was sufficient for malignant transformation. CBFA2T3-GLIS2 positive cases displayed marked upregulation of genes with cell membrane/extracellular matrix localization potential, including NCAM1 and GABRE. Additionally, miRNA profiling revealed significant overexpression of mature miR-224 and miR-452, which are intronic miRNAs transcribed from the GABRE locus. Gene-set enrichment identified dysregulated Hippo, TGFb, and hedgehog signaling, as well as NCAM1 (CD56) interaction pathways. Therapeutic targeting of fusionpositive leukemic cells with CD56-directed antibody-drug conjugate caused significant cytotoxicity in leukemic blasts. Conclusions: The CBFA2T3-GLIS2 fusion defines a highly refractory entity limited to infants that appears to be sufficient for malignant transformation. Transcriptome profiling elucidated several highly targetable genes and pathways, including the identification of CD56, providing a highly plausible target for therapeutic intervention.

AID (Activation Induced Deaminase) deaminates cytosines in DNA to initiate immunoglobulin gene diversification and to reprogram CpG methylation in early development. AID is potentially highly mutagenic, and it causes genomic instability evident as translocations in B cell malignancies. Here we show that AID is cell cycle regulated. By high content screening microscopy, we demonstrate that AID undergoes nuclear degradation more slowly in G1 phase than in S or G2-M phase, and that mutations that affect regulatory phosphorylation or catalytic activity can alter AID stability and abundance. We directly test the role of cell cycle regulation by fusing AID to tags that destabilize nuclear protein outside of G1 or S-G2/M phases. We show that enforced nuclear localization of AID in G1 phase accelerates somatic hypermutation and class switch recombination, and is well-tolerated; while nuclear AID compromises viability in S-G2/M phase cells. We identify AID derivatives that accelerate somatic hypermutation with minimal impact on viability, which will be useful tools for engineering genes and proteins by iterative mutagenesis and selection. Our results further suggest that use of cell cycle tags to regulate nuclear stability may be generally applicable to studying DNA repair and to engineering the genome.

In an effort to identify acute myeloid leukemia (AML)-restricted targets for therapeutic development in AML, we analyzed the transcriptomes of 2051 children and young adults with AML and compared the expression profile with normal marrow specimens. This analysis identified a large cohort of AML-restricted genes with high expression in AML, but low to no expression in normal hematopoiesis. Mesothelin (MSLN), a known therapeutic target in solid tumors, was shown to be highly overexpressed in 36% of the AML cohort (range, 5-1077.6 transcripts per million [TPM]) and virtually absent in normal marrow (range, 0.1-10.7 TPM). We verified MSLN transcript expression by quantitative reverse transcription polymerase chain reaction, confirmed cell surface protein expression on leukemic blasts by multidimensional flow cytometry, and demonstrated that MSLN expression was associated with promoter hypomethylation. MSLN was highly expressed in patients with KMT2A rearrangements (P &lt; .001), core-binding factor fusions [inv(16)/t(16;16), P &lt; .001; t(8;21), P &lt; .001], and extramedullary disease (P = .001). We also demonstrated the presence of soluble MSLN in diagnostic serum specimens using an MSLN-directed enzyme-linked immunosorbent assay. In vitro and in vivo preclinical efficacy of the MSLN-directed antibody-drug conjugates (ADCs) anetumab ravtansine and anti-MSLN–DGN462 were evaluated in MSLN+ leukemia cell lines in vitro and in vivo, as well as in patient-derived xenografts. Treatment with ADCs resulted in potent target-dependent cytotoxicity in MSLN+ AML. In this study, we demonstrate that MSLN is expressed in a significant proportion of patients with AML and holds significant promise as a diagnostic and therapeutic target in AML, and that MSLN-directed therapeutic strategies, including ADCs, warrant further clinical investigation.

The CBFA2T3-GLIS2 (C/G) fusion is a product of a cryptic translocation primarily seen in infants and early childhood and is associated with dismal outcome. Here, we demonstrate that the expression of the C/G oncogenic fusion protein promotes the transformation of human cord blood hematopoietic stem and progenitor cells (CB HSPCs) in an endothelial cell coculture system that recapitulates the transcriptome, morphology, and immunophenotype of C/G acute myeloid leukemia (AML) and induces highly aggressive leukemia in xenograft models. Interrogating the transcriptome of C/G-CB cells and primary C/G AML identified a library of C/G-fusion-specific genes that are potential targets for therapy. We developed chimeric antigen receptor (CAR) T cells directed against one of the targets, folate receptor α (FOLR1), and demonstrated their preclinical efficacy against C/G AML using in vitro and xenograft models. FOLR1 is also expressed in renal and pulmonary epithelium, raising concerns for toxicity that must be addressed for the clinical application of this therapy. Our findings underscore the role of the endothelial niche in promoting leukemic transformation of C/G-transduced CB HSPCs. Furthermore, this work has broad implications for studies of leukemogenesis applicable to a variety of oncogenic fusion-driven pediatric leukemias, providing a robust and tractable model system to characterize the molecular mechanisms of leukemogenesis and identify biomarkers for disease diagnosis and targets for therapy.

has equity ownership in Hematologics, Inc. S.F. holds provisional patents filed on the use of CAR-T cell therapy for hematologic malignancies and is a medical and scientific advisor for Link Immunotherapeutics. C.J.T receives research funding from Juno Therapeutics/BMS, Nektar Therapeutics, Minerva, AstraZeneca, TCR 2 Therapeutics; is a member of scientific advisory boards for Precision Biosciences, Eureka Therapeutics, Caribou Biosciences, T-CURX, Myeloid Therapeutics, ArsenalBio, and Century Therapeutics; has served on an ad hoc advisory board (last 12 months) for Amgen; holds stock options in Precision Biosciences, Eureka Therapeutics, Caribou Biosciences, Myeloid Therapeutics, and ArsenalBio; and receives royalties for unrelated patents licensed to Juno Therapeutics/BMS. All other authors declare no competing financial interests.Research.

Fusion oncoproteins are the initiating event in AML pathogenesis, although they are thought to require additional cooperating mutations for leukemic transformation. CBFA2T3-GLIS2 (C/G) fusion occurs exclusively in infants and is associated with highly aggressive disease1-4. Here we report that lentiviral transduction of C/G fusion is sufficient to induce malignant transformation of human cord blood hematopoietic stem and progenitor cells (CB HSPCs) that fully recapitulates C/G AML. Engineered CB HSPCs co-cultured with endothelial cells undergo complete malignant transformation with identical molecular, morphologic, phenotypic and disease characteristics observed in primary C/G AML. Interrogating the transcriptome of engineered cells identified a library of C/G fusion-specific targets that are candidates for chimeric antigen receptor (CAR) T cell therapy. We developed CAR-T cells directed against one of the targets, FOLR1, and demonstrated the pre-clinical efficacy against C/G AML while sparing normal hematopoiesis. Our findings underscore the role of the endothelial niche in promoting leukemic transformation of C/G-transduced CB HSPCs. Moreover, this work has broad implications for studies of leukemogenesis applicable to a variety of oncogenic fusion-driven pediatric leukemias, providing a robust and tractable model system to characterize the molecular mechanisms of leukemogenesis and identify biomarkers for disease diagnosis and targets for therapy.

E-selectin (E-sel) is a cell adhesion glycoprotein that is expressed on endothelial cells and has been implicated in therapeutic resistance. In most myeloid leukemias, leukemic blasts express E-sel ligands (EsL), which contain the glycan epitope of the carbohydrate sialyl Lex (sLex). This expression increases the likelihood of adhesion to vascular endothelial cells and facilitates sequestration in the bone marrow vascular niche, leading to cell adhesion-mediated drug resistance and poor clinical outcome. E-sel antagonists like uproleselan, interrupts leukemic cell homing to the vascular niche, increases susceptibility to cytotoxic and targeted therapies and can be potent adjuncts to therapeutics. Recent data demonstrated a correlation between leukemic cell surface levels of EsL and response to uproleselan, linking EsL expression to response. We questioned whether transcriptome profiling of EsL-forming glycosylation genes can be used to identify elevated EsL expression in patients with acute myeloid leukemia (AML), and subsequently which patients might best respond to uproleselan. RNA-seq data from patients treated in COG AAML1031 (N = 1,074) was available for evaluation. We examined transcriptome expression of 24 genes that code for enzymes involved in glycosylation of EsL. All analyses were performed in R. Cox proportional hazards models were generated using the survival package. Multidimensional flow cytometry (MDF) was used to detect cell surface EsL expression by two techniques: direct binding of an E-sel/hIg, PE labeled chimera, and the anti-sLex antibody HECA-452. Seven of the 24 genes examined had minimal expression (mean <1 TPM) and were excluded from further analysis. The remaining 17 were variably expressed (Fig. 1A). To assess association of expression with outcome, univariate Cox models for overall survival (OS) were generated, using gene expression as a continuous coefficient (N = 1,061). Of the 17 genes, 7 were significantly associated with increased risk (p < 0.05, Fig. 1B). ST3GAL4 and FUT7 were targeted for further evaluation, as they directly synthesize sLex (Fig. 1C), and were significantly associated with adverse outcome (HR = 1.013, p < 0.0001, and HR = 1.023, p < 0.0001, respectively). Patients highly expressing FUT7 (highest quartile of expression) had significantly worse outcome than low expressors (lowest 3 quartiles of expression), with a 5-year OS of 50.3% vs. 68.3% (p < 0.0001, Fig. 1D). Similarly, those with high ST3GAL4 expression had a 5-year OS of 51.3%, compared to 68.1% for low expressors (p < 0.0001, Fig. 1D). A subset of patients highly expressed both genes (ST3GAL4 and FUT7 high; SFhigh, N = 132). Compared to patients that did not highly express either gene (SFlow), these individuals had particularly adverse survival (45.8% OS vs 71.0% OS, p < 0.0001). Patients with one of two high expressing genes (SFinter) had a 5-year OS of 55.5%, illustrating what may be a compounding unfavorable impact conferred on survival (Fig. 1E). Further investigation of clinical characteristics within these 3 groups revealed that 71.5% of infants <1 year were SFlow, with only 4.66% in SFhigh. In addition, CBF-AML was greatly underrepresented in SFhigh, with 97% of both t(8;21) and inv(16) patients in SFlow, and 0% in SFhigh. To verify surface protein expression of the two genes, leukemic specimens from SFhigh patients (N = 10) and SFlow patients (N = 10) underwent cell surface expression evaluation of glycosylated EsL using two MDF assays. SFlow patients had low or undetectable levels of cell surface EsL by both assays, whereas SFhigh patients had significantly higher expression of EsL (p < 0.001, Fig. 1F). This suggests a strong correlation between transcriptome measurements of EsL glycosylation genes and cell surface glycosylation levels of EsL. In summary, we have shown that multiple genes involved in the glycan synthesis of EsLs are highly expressed in pediatric AML. Two of these genes, ST3GAL4 and FUT7, are associated with poor outcome. Additionally, high expression of these genes is detectable at the transcript level and associated with cell surface EsL expression. These genes provide novel targets for overcoming drug resistance induced by the tumor microenvironment, and lend support for the use of EsL glycosylation genes as predictive biomarkers. These data also confirm the importance of E-sel in disease progression in AML and its potential as a therapeutic target. Figure 1 Disclosures Pardo: Hematologics, Inc: Employment. Eidenschink Brodersen:Hematologics, Inc: Employment. Magnani:GlycoMimetics Inc: Employment, Equity Ownership. Fogler:GlycoMimetics Inc: Employment, Equity Ownership. Loken:Hematologics, Inc: Employment, Equity Ownership.

Preferentially Expressed Antigen in Melanoma (PRAME) , a cancer testes antigen provides an ideal target for immunotherapy in AML. We have shown expression of PRAME in a significant subset of childhood and adult AML and lack of expression in normal hematopoiesis. Although an intracellular antigen, we developed a novel approach to target PRAME using a CAR construct encoding a targeting domain based on T cell receptor (TCR) mimic antibodies that targets the peptide:HLA complex. We used the antibody sequence from a previously designed antibody, Pr20, a TCR mimic antibody that recognizes PRAME ALY peptide in complex with HLA-A*02 and verified expression of PRAME in AML cell lines and primary AML blasts. Using the Pr20 antibody sequence, we developed CAR T cells (PRAME mTCRCAR T) to be tested against primary AML patient samples and AML cell lines that express the PRAME antigen in the context of HLA-A2 expression. In contrast to the appropriate controls, PRAME mTCRCAR T cells demonstrate target specific and HLA-mediated in vitro activity in OCI-AML2 and THP-1 cell lines, HLA-A2 cell lines expressing the PRAME antigen, and against primary AML patient samples. In vivo cell-derived xenograft models treated with PRAME mTCRCAR T cells demonstrated potent leukemia clearance and improved survival compared to unmodified T-cell controls. Furthermore, the cytolytic activity of PRAME mTCRCAR T cells was enhanced by treating the target cells with IFN-γ, which increases PRAME antigen expression. These results demonstrate the feasibility and efficacy of targeting PRAME with novel PRAME mTCRCAR T cells.