Mesothelin Is a Novel Disease Marker and Potential Therapeutic Target in Pediatric Acute Myeloid Leukemia

Acute myeloid leukemia (AML) remains a significant therapeutic challenge in children with only 50% cure rate with highly intensive cytotoxic chemotherapy. Further intensification is limited by acute and late effects, including severe infectious and cardiac toxicities; therefore, alternative therapeutic strategies are needed. As part of the NCI/COG TARGET AML Initiative, using transcriptome sequencing (RNA Seq) in 635 children and 178 adult AML, we demonstrated that mesothelin (MSLN) was highly-overexpressed in over one-third of pediatric AML. This transcript is not expressed in normal hematopoiesis, making it an ideal target for therapeutic intervention. MSLN is a cell-surface protein normally found in pleura and peritoneum and overexpressed in a variety of solid tumors, such as mesothelioma. The protein product of the MSLN gene exists in 4 forms in the body, including cell surface MSLN, soluble MSLN, and soluble megakaryocyte potentiating factor (MPF), which is the N-terminus of the MSLN pro-protein. Elevated serum soluble MSLN is an established diagnostic test for mesothelioma, with an FDA-approved ELISA-based assay (Mesomark™).

Three different AML RNA Seq data sets totaling 813 cases, including TARGET validation cohort (n= 475), TARGET discovery phase (n= 160), and TCGA AML (n= 178), were interrogated for expression of MSLN, with normal marrow cases as a control. MSLN transcript expression at >5 transcripts per million (TPM) was observed in 268 patients (range 0-1225 TPM) with observed prevalence of 48% in TARGET discovery set, 33% in TARGET validation and 15% in adult TCGA set with a collective prevalence of 33% in the entire cohort. MSLN expression was enriched in patients with t(8;21), inv(16), and KMT2A fusions and rare in those with common AML-associated mutations (FLT3- ITD, NPM1, CEBPa mutations). MSLN was not expressed in 20 normal marrow samples (<1 TPM in all cases).

We conducted orthogonal validation of the transcriptome data by evaluating diagnostic specimens from an independent cohort of 323 newly diagnosed pediatric AML patients for the presence of MSLN using quantitative RT-PCR and ELISA. RNA extracted from leukemic blasts or plasma obtained from diagnostic specimens were tested for MSLN expression by quantitative RT-PCR or sandwich ELISA, respectively. Optimized q-RT-PCR identified 36% of patients with MSLN expression. Similarly, sandwich ELISA (similar to Mesomark™) confirmed expression of soluble MSLN in 34% of patients tested, with strong concordance between the two tests (p <0.0001). We have also optimized diagnostic multi-dimensional flow cytometry (MDF) and MPF ELISA for evaluation of MSLN.

Mesothelin is a cell surface protein highly expressed in AML blasts but absent in other hematopoietic cells, making it an attractive target for immunotherapy. We sought to test the principle of MSLN targeting in leukemia with antibody-drug conjugate (ADC) anetumab ravtansine (AR; BAY94-9343), comprised of an anti-MSLN antibody conjugated to tubulin inhibitor DM4. MSLN -overexpressing cells were created by lentivirally-transducing MSLN into K562 cells (K562+ MSLN). K562+ MSLN exhibited exquisite sensitivity to AR in vitro with an IC₅₀ of 1.4 nM, compared to 360 nM in the non-transduced K562 and 250-260 nM in isotype control conditions (Fig. 1). In vitro cytotoxicity of AR in two primary pediatric MSLN + AML samples also demonstrated MSLN -dependent killing. Additionally, mice bearing K562+ MSLN xenografts were treated in vivo with AR, which demonstrated significant MSLN -dependent efficacy, resulting in median survival of 87 days compared to 32-41 days in control conditions, including chemotherapy with daunorubicin and cytarabine (Fig. 2).

MSLN is a cell-surface protein uniquely overexpressed in one-third of pediatric AML, as well as a subset of adult AML. We have shown that MSLN can be detected through several sensitive diagnostic mechanisms including MDF, q-RT-PCR and serum soluble MSLN, making it an optimal marker for disease monitoring. The leukemic expression of MSLN and absence from normal hematopoietic tissue make it an ideal target for immunotherapeutic targeting, and our findings demonstrate that MSLN can be exploited for therapeutic benefit using ADCs. Further studies in MSLN + AML on optimal detection and immunotherapeutic strategies are needed, as MSLN shows great promise for enhanced disease monitoring and leukemia-specific therapeutic targeting.

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