Aspartate β-hydroxylase (ASPH) is an α-ketoglutarate-dependent dioxygenase that promotes cellular growth, motility and adhesion by post-translational hydroxylation of aspartyl and asparaginyl residues in epidermal growth factor-like protein domains, including Notch, Notch homologs, Jagged and extracellular matrix molecules such as laminin and tenascin (1, 2). ASPH is encoded by the aspartate beta-hydroxylase (ASPH) gene on chromosome 8 and is upregulated by insulin and insulin growth factor 1 (IGF1) through the MAPK/ERK and PI3K/AKT pathways (2, 3). ASPH can lead to carcinogenesis by inducing decreased cleavage of caspase-3, causing inhibition of apoptosis (4), and may also promote tumor immune escape via inhibition of natural killer (NK)-cell activity (5).

ASPH is highly expressed during fetal development and is aberrantly upregulated in cancer cells (6). ASPH is overexpressed in over 20 different solid neoplasms, including liver (7–10), breast (11), lung (12), brain (13), pancreatic (14), gastric (15) and colorectal cancers (16), in which it propagates a malignant phenotype, associated with increased cell proliferation, invasiveness, metastasis, and also with poor clinical prognosis (7, 8, 17–19).

ASPH has been shown to be immunogenic in preclinical studies exploring its role as a target for vaccination (20, 21) and for dendritic cell therapy (22), making it a promising immunotherapeutic target (23–25). ASPH is currently being targeted with an anti-ASPH nanoparticle vaccine, SNS-301, in a clinical trial in prostate cancer (26).

Despite the established role of ASPH in solid neoplasms, little is known about its role in hematologic malignancies. Acute myeloid leukemia (AML) is a heterogeneous hematologic malignancy with an incidence of 4.3 per 100,000 in the US and a 5-year survival rate of only 24% (27). New treatment options are needed for AML and much interest has shifted toward immunotherapeutic strategies in treatment of AML, necessitating identification of leukemia-associated antigens. Studies have demonstrated overexpression of ASPH in the AML cell line MOLM-14 in vitro, and successful targeting of myeloblasts with anti-ASPH radiolabeled or cytotoxin-linked antibody drug-conjugates (ADCs) (28, 29).

ASPH may have the potential to serve as a tumor- or leukemia-associated antigen for immunotherapy. In an effort to further characterize ASPH as a therapeutic target in AML, we report here the first study of ASPH expression in AML patient samples.

ASPH was overexpressed on blasts of approximately 40% of AML patients in this series. While not appearing to be associated with any specific disease features or with clinical prognosis, this surface expression serves as a potential new therapeutic target for AML.

Discovery of new therapeutic targets in AML remains imperative. Specifically, new treatments are needed for patients with relapsed/refractory disease or those who are unfit for intensive cytotoxic chemotherapy. Immunotherapeutic strategies using ADCs, bispecific T-cell engagers (BiTEs), and chimeric antigen receptor (CAR) T-cell therapy have shown great success in B-cell lymphoid leukemia and lymphoma due to the consistently expressed, safe and effective targets CD19 and/or CD22 (31–34). However, progress for AML has lagged because of lack of a selective leukemia-associated target. Therefore, much interest has been focused on discovery of new tumor- or leukemia-associated antigens, such as ASPH.

ASPH is a safe therapeutic target due to its inherent embryonic function, which is not required for healthy adult human function (barring its possible role in pregnancy) (35, 36). Mutations in the ASPH gene lead to Traboulsi Syndrome in humans, reported in four families in the literature. This syndrome is characterized by developmental defects including facial dysmorphism and ophthalmologic abnormalities such as lens dislocation and anterior-segment abnormalities. Most importantly, monitoring of these patients has shown that the loss of ASPH function has not been associated with disease later in life (37).

ASPH is further a promising target due to its immunogenicity. When expressed, ASPH is found at high levels on the cell surface plasma membrane, where its N-terminal sequences and the catalytic site located in the C-terminal region have been shown to serve as antigens that are accessible to the immune system (21). In preclinical studies, vaccination is capable of inducing ASPH epitope-specific CD4⁺ and CD8⁺ T-cell responses in both animal and human models of hepatocellular carcinoma (26). Dendritic cell vaccination studies in a rodent model of biliary cancer showed successful passive vaccination with naïve dendritic cells that were matured in vivo in the presence of ASPH, leading to tumor regression, along with ASPH-specific T-cell responses (25). ASPH is also thought to lead to immune surveillance escape and promotes tumor growth by direct effects on NK-cells, including reducing NK-cell viability and cytotoxicity (5).

Importantly, unlike data in solid tumors that suggest the role of ASPH as a predictive marker of poor clinical prognosis (17–19), we found that ASPH status was not associated with clinical outcomes (CR, EFS, OS) in AML. Further, there was no association seen between ASPH status and cytogenetic abnormality, mutation status, or ELN risk category, though notably the majority (74%) of our patient cohort fell into the unfavorable risk category. This may reflect the limitations of our study, including the relatively small patient cohort and the retrospective nature of our clinical outcome review, along with the significant heterogeneity of AML as a clinical entity. The heterogeneity of AML as seen by various combinations of cytogenetic and molecular characteristics that define disease subsets and control how aggressive the disease can be, is important to consider, as due to the small cohort in this study, this heterogeneity may explain the lack of association found with any significant outcome and begs for further large scale studies. Also, changes can be perhaps seen longitudinally in serial samples of the same patient after treatment, where AML myeloblasts can exhibit changes to surface antigen expression and clonal evolution at times, which would be another interesting angle to explore in future trials and not represented by our study that only included samples at time of diagnosis. Additionally, a further limitation may be that while CR data was available for all patients, measurable residual disease (MRD) status at time of CR based on molecular next-generation sequencing was not available for all given the time period of sample collection, which was partly in the years prior to increasing standardization of MRD and its currently established influence on clinical outcomes.

One hypothesis is that while ASPH plays an important role in cancer cell metastasis in solid neoplasms, including cellular detachment, migration, and adhesion at a distant site (19), circulating tumor cells such as leukemic blasts do not need a seeding advantage in order to exert their deleterious effects that lead to poor prognosis. Further investigation regarding a possible relationship between ASPH expression and extramedullary disease in AML would be interesting to explore in future studies. While higher expression of ASPH has been reported in some cancer stem cells such as glioma stem cells (38), its expression on leukemia stem cells (LSC) is not well described. Another question of interest in study of ASPH in leukemia, specifically, includes whether ASPH is differentially expressed in leukemia stem cells compared to more differentiated leukemia blasts, and while this study did not address this question – there has been much study into genes that play a role in stemness for AML. One such study pursuing the development of predictive and/or prognostic biomarkers related to stemness, Ng et al. generated a list of genes that are differentially expressed between 138 LSC positive (LSC+) and 89 LSC− cell fractions from 78 AML patients validated by xenotransplantation. They generated a 17-gene LSC score (LSC17). ASPH was not among the 17 signature genes, which were GPR56, AKR1C3, CD34, NGFRAP1, EMP1, SMIM24, SOCS2, CPXM1, CDK6, KIAA0125, DPYSL3, MMRN1, LAPTM4B, ARHGAP22, NYNRIN, ZBTB46, and DNMT3B. Of note, the LSC17 score was highly prognostic in five independent cohorts of approximately 900 AML patients (39).

An additional potentially important finding was the statistically significant higher incidence of ASPH expression on blasts of African American AML patients. It is unknown if this is a pattern seen across other cancers with ASPH expression. Though our relatively small cohort is a limiting factor, this finding should be further explored prospectively and validated. Racial disparities and their effect on clinical outcomes for AML represent an important field of interest that requires more investigation (40, 41).

Lastly, it has been reported that embryonic antigens, such as ASPH, are upregulated by HMAs (42–44). In our patient cohort, approximately one third (34%) of patients who were treated with HMA (alone or in combination with another drug) had ASPH-positive AML at diagnosis. Among these 14 patients, 4 (29%) were secondary AML patients, 4 (21%) were relapsed/refractory AML, and 7 (50%) were newly diagnosed AML. Importantly, for all these patients, the HMA was administered after the collection of the sample that was analyzed for ASPH expression – none received HMA prior. While the decision to use HMAs likely reflects patients’ older age, poor performance status or AML features, such as complex karyotype, HMA use and potential effects on ASPH expression warrant further investigation. Future studies exploring ASPH expression on serial samples in patients who were treated with or without HMAs would be of great interest. Further, there is potential for synergy when using HMAs as they may increase ASPH antigen expression to allow for concurrent targeting of surface ASPH expression with an immunotherapeutic agent.

In summary, ASPH is overexpressed in approximately 40% of AML cases, and can serve as a potential immunotherapeutically targetable tumor-specific antigen. An anti-ASPH nanoparticle vaccine is currently under clinical investigation, having completed Phase 1 testing with encouraging results in solid tumors (29). Additional immunotherapies such as ADCs and CAR T-cells targeting ASPH may be promising potential therapeutic agents for AML.

The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.

The studies involving human participants were reviewed and approved by University of Maryland School of Medicine (UMSOM) Institutional Review Board (IRB). The patients/participants provided their written informed consent to participate in this study.

Conception: NH, AE, ML, and HG. Interpretation or analysis of data: NH, ML, AE, RK, MB, and SB. Manuscript preparation: NH, AE, ML, RK, MB, KM, AS, HG, and SB. Revision for important intellectual content: NH, AE, ML, RK, MB, and HG. Supervision: AE, ML, RK, and KM. All authors contributed to the article and approved the submitted version.

This research was supported by funds through the National Cancer Institute–Cancer Center Support Grant (CCSG)–P30CA134274.

Authors ML, KM and AS were employed by company Sensei Biotherapeutics.

ML: Stock ownership in Sensei. AS: Stock ownership in and received consultancy fee from Sensei. HG: Founder and emeritus CSO, Panacea Pharmaceuticals/Sensei Biotherapeutics, Founder/CEO, Athanor Biosciences, MD.

The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.