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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 < .001), core-binding factor fusions [inv(16)/t(16;16), P < .001; t(8;21), P < .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.

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In efforts to discover genes uniquely expressed in childhood AML, we performed transcriptomesequencing (RNA-Seq) in pediatric AML and contrasted the expression signature to that in normal marrow hematopoiesis. This effort led to the discovery of over 200 genes that lack expression in normal hematopoietic cells, but are variably expressed in pediatric AML cells. Mesothelin(MSLN) was discovered to be one of the most highly expressed genes in a subset of childhood AML cases (p<10-15).Mesothelin is a cell-surface protein that is expressed onmesothelial cells ofserosal lining. MSLN is over-expressed on a variety of solid tumors, including lung, pancreatic, and ovarian cancers, and is associated with increased malignant transformation, cellular proliferation, and tumor aggressiveness. Given its cell surface expression, MSLN has emerged as an attractive target for immunotherapeutic interventions in solid tumors in adults. In this study, RNA obtained from diagnostic bone marrow specimens from childhood AML (N=434) and normal marrow (N=20) was subjected to wholetranscriptomesequencing and MSLN expression was quantified and normalized and reported as reads perkilobaseof exon per million reads mapped (RPKM). Similar data was obtained from adult TCGA AML database. Quantitative RT-PCR (qRT-PCR) and multidimensional flowcytometry(MDF) was used for confirmation of the transcript and cell surface protein expression. TARGET AML methylation data was used for correlation with transcript expression. Of the 434 specimens analyzed, MSLN mRNA expression was variably expressed (RPKM range 0-618.8), with expression detected in 119 patients (27%). Confirmatory studies by qRT-PCR on specimens with and without MSLN expression (N=137) showed correlation between RNA-Seqand PCR data. Cell surface MSLN expression was assessed by MDF using a PE-conjugated MSLN antibody (PE-mesoAb) and verified expression of MSLN protein on the leukemic cell surface in every case with MSLN transcript expression (Figure 1A). Evaluation of CD34+/CD38- hematopoietic progenitor cells by PE-mesoAbdemonstrated lack of MSLN expression by MDF. Evaluation of matched diagnostic and relapse specimens from MSLN-expressing patients (n=27) confirmed that MSLN expression was largely stable (R2=0.87), thus substantiating its expression in the major AML clone. Comparison of MSLN expression in pediatric vs. adult AML demonstrated a higher prevalence in pediatric AML (TARGET: 27% vs. TCGA: 11%)(Figure 1B). Evaluation of the clinical and biologic features in MSLN expressing (MSLN+) and non-MSLN expressing (MSLN-) pediatric patients revealed that MSLN expression was rarely observed in patients with normal karyotype (p<0.001) or with the most common somatic mutations of FLT3/ITD, NPM1, CEBPA (p<0.001 in all cases). However, MSLN expression was significantly higher in patients with inv(16), t(8;21) and MLL translocations (p<0.001, p<0.001, and p=0.02 respectively; Figure 1C). Given that a majority of patients with core binding factor (CBF) AML were MSLN+, we evaluated the clinical implications of MSLN expression in this favorable risk cohort. Among CBF patients, MLSN+ patients (n=95) had a relapse risk of 51% vs. 32% in the MSLN- (n=62) cohort (p=0.03; Figure 1D), with a corresponding disease free survival of 46% vs. 64% respectively (p=0.03). We further inquired about the mechanism by which MSLN expression might be regulated. Whole genome sequencing data failed to identify any genomic alterations in MSLN that could result in high expression. Therefore, we interrogated the possibility of epigenetic regulation of MSLN expression. Integration of the expression and methylation profiling cases with matching RNA-Seqand methylation data (N=246) demonstrated thathypomethylationof the MSLN promoter significantly correlated with high MSLN expression, implicating epigenetic regulation in the expression of MSLN in AML. Mesothelin, a therapeutic target in solid tumors, is highly expressed in biologically distinct subsets of childhood AML. High expression on leukemic blasts and lack of expression in normal hematopoiesis makes this antigen an ideal target for therapeutic intervention in AML. As a cell surface protein, this antigen avails itself for immune targeting by antibody drug conjugates, CAR-T cells, and T cell receptor mediated targeting. Figure 1 Mesothelin expression in childhood AML. Figure 1. Mesothelin expression in childhood AML. Disclosures Loken: Hematologics: Employment, Equity Ownership. Pardo:Hematologics, Inc: Employment.