DYRK1A controls CD47 endocytic degradation and leukemia immune surveillance Free

AML is an aggressive blood cancer with no effective immunotherapies. One mechanism by which AML evades immune surveillance is by upregulating the macrophage checkpoint CD47 and thus resisting phagocytosis. However, initial clinical efforts to block CD47 via antibodies were complicated by widespread CD47 expression in normal hematopoietic cells and did not improve outcomes. As such, identifying CD47's regulatory mechanisms in AML versus normal cells could have significant therapeutic implications.

To identify regulators of cell surface CD47, we performed CD47 antigen-guided genome-wide CRISPR knockout (KO) screens in MOLM-13, THP-1, and MV4;11 AML cell lines in vitro and in vivo. One consistent finding in vitro and in vivo across all cell lines was that KO of the Serine/Threonine kinase DYRK1A reduced cell surface CD47. DYRK1A KO promoted phagocytosis of AML cells when co-cultured with mouse macrophages in vitro, inhibited the growth of AML in vivo, and prolonged the survival of AML-bearing NSG mice. Conversely, overexpression of DYRK1A or its paralog DYRK1B increased cell surface CD47 abundance in AML, in a manner dependent on DYRK kinase activity.

As DYRK1A is implicated in diabetes, neurodegenerative diseases, and neurologic and malignant complications of Down syndrome (given DYRK1A's location on chromosome 21), there is significant interest in developing selective DYRK1A inhibitors. Additionally, amongst the TCGA profiled malignancies, DYRK1A mRNA expression is most highly upregulated in AML. However, previously described DYRK1A inhibitors also inhibit the closely related CLK kinases and/or GSK3β with similar potency. SM13797, a novel ultra-selective DYRK1A inhibitor was therefore developed by Biosplice utilizing structure-based drug design and iterative medicinal chemistry optimization. In vitro kinase assays and cell based NanoBRET competition assays demonstrated exquisite potency and selectivity of SM13797 for DYRK1A/B compared to previously described DYRK inhibitors such as Harmine, EHT1610, and GNF2133.

SM13797 reduced cell surface CD47 across AML cell lines in a dose-dependent manner. Furthermore, treatment of MV4;11 AML xenograft mice with SM13797 reduced cell surface CD47 abundance and increased phagocytosis of AML cells by mouse macrophages in vivo. Long-term SM13797 treatment in vivo increased mouse F4/80⁺ phagocyte infiltration and reduced human AML burden, yielding a median survival benefit of 30 days. Importantly, pre-treatment of animals with clodronate liposomes to eliminate macrophages revealed that macrophages are required for therapeutic efficacy of selective DYRK1A inhibition.

To understand the mechanism by which DYRK1A regulates CD47, we performed serine/threonine phosphoproteomics on MV4;11 and THP-1 AML cells treated with SM13797. DYRK1A substrates in AML were enriched in proteins involved in clathrin-mediated endocytosis, such as CBL, SYNJ1, and REPS1. Knockout of each of these factors attenuated the reduction in cell surface CD47 seen with SM13797. Consistently, SM13797 promoted the formation of endolysosomal compartments based on electron microscopy, and enhanced CD47 endocytosis in a lysosome-dependent manner in AML cells.

Given that the E3 ubiquitin ligase CBL is frequently mutated in myeloid neoplasms, we dissected the connection between DYRK1A and CBL further. SM13797 inhibited CBL phosphorylation at Serine 452 (S452) and increased CBL protein levels by influencing ubiquitination at the adjacent Lysine 424 residue. We validated these findings further using AML cells expressing CBL S452 phospho-defective or -mimetic mutants and subjecting them to cycloheximide chase experiments. Leukemia-associated CBL mutants (defective in E3 ligase functions) reduced CD47 endocytosis and increased cell surface CD47 abundance. CD47 immunoprecipitation-western blot validated the K63-linked polyubiquitination of CD47 by CBL. As such, DYRK1A-mediated regulation of CBL in turn influences CD47 endocytosis.

This study discovers a role for DYRK1A in innate immune recognition via regulation of CD47. Genetic or pharmacologic inhibition of DYRK1A in AML disrupts DYRK1A-mediated phosphorylation of endocytic regulators such as CBL, thereby promoting CD47 endocytosis, reducing cell surface CD47 abundance, and enhancing macrophage-mediated phagocytosis. Finally, we identify a novel ultra-selective DYRK1A inhibitor whose promising preclinical activity in AML harnesses innate immune recognition.