A PDZ-Binding Motif in DCAF7 Maintains Hematopoietic Homeostasis and Prevents Malignancy in DS-AMKL
Authors: Kirsammer Gina , Schultz Rachael F , Rastogi Ritika , Gurbuxani Sandeep , Crispino John 
Published: November 2019

Abstract
Down syndrome-associated acute megakaryoblastic leukemia (DS-AMKL) affects 1 in 500 children with DS and associates uniformly with somatic mutations in GATA1. DS-AMKL is often treated successfully; however, treatments can cause long-term side effects, such that mechanistic insights and targeted therapies are needed.
Recent studies report a recurring L340F mutation in DCAF7 in DS-AMKL, at the C-terminus of the protein. A highly conserved scaffold protein, DCAF7 is a cofactor for the chromosome 21-encoded kinase, DYRK1A, and shares 100% protein sequence homology among vertebrates. In our efforts to characterize the normal function of DCAF7 in hematopoiesis and its contribution to AMKL, we have identified an unreported PDZ-binding motif (PBM) in DCAF7, which is perturbed by the L340F mutation. Overexpression of mutant (DCAF7mut), but not wild type DCAF7 (DCAF7wt), in a mouse model of DS with a Gata1 mutation leads to rapid onset of dysplastic disease characterized by peripheral blood leukopenia and thrombocytopenia, with normal red cell numbers. Bone marrow of affected mice harbors abnormal, hyperproliferative erythroid progenitors and diminished, hyperchromatic megakaryocytes, with stark depletion of granulocytic and lymphoid lineages and widespread edema. Splenic architecture is grossly abnormal, with a complete absence of white pulp, proliferation of erythroid precursors, and significant numbers of apoptotic, hyperchromatic megakaryocytes.
Conversely, targeted deletion of Dcaf7 in hematopoiesis shifts the myeloid-erythroid balance toward the myeloid lineage, with increased granulopoiesis and hyperchromatic megakaryocytes in the bone marrow and lower white blood cell numbers and dramatic thrombocytopenia in the peripheral blood. Splenic architecture is largely normal, although flow cytometry reveals defective B- and T- cell development.
The bone marrow knockout phenotype of Dcaf7 is strongly reminiscent of the cellular phase of primary myelofibrosis (PMF). Combined with bone marrow overexpression phenotypes, which resemble hemophagocytic syndrome, our data suggest that DCAF7 is critically important for maintaining a healthy bone marrow environment and regulating blood cell lineage choice. In order to identify functional binding partners of the DCAF7 PDZ-binding motif (PBM) that may support these functions, we performed IP mass spectrometry using DCAF7wt or DCAF7mut as bait and found two highly conserved PDZ-domain containing scaffold proteins, PAR3 and PDLIM5, that interact robustly with DCAF7wt, but not with DCAF7mut. While both DCAF7wt and DCAF7mut are able to associate with DYRK1A, the ability of DCAF7 to bring DYRK1A activity to the PAR3 and PDLIM5 signaling hubs affects the composition of DCAF7-scaffolded transcriptional complexes and downstream transcriptional outcomes.
Through RNA-sequencing and Ingenuity Pathways Analysis, overexpression of both DCAF7wt and DCAF7mut in mouse bone marrow leads to the induction of interferon response genes; however, DCAF7wt also induces a pro-apoptotic gene program, whereas DCAF7mut instead promotes survival gene expression and developmental reprogramming. Consistently, expression of DCAF7wt in bone marrow progenitors causes rapid expansion and increased initial colony forming ability, but decreased replating potential compared with vector control. Conversely, expression of DCAF7mut causes increased proliferation and colony formation accompanied by enhanced replating, indicating greater self-renewal conferred by DCAF7mu.
Our data indicate DCAF7 regulates the balance of proliferation, differentiation and apoptosis in hematopoietic cells and that the PBD of DCAF7 is critically important for these functions, connecting DCAF7 to signaling hubs that translate environmental cues into appropriate transcriptional outcomes. Furthermore, DCAF7 disruption causes hematopoietic disease reminiscent of human pathology, suggesting it may have broad significance in hematopoietic malignancy. Finally, our data support a model wherein DCAF7 mutation facilitates the development of malignancy by promoting the survival and proliferation of mutant clones in the bone marrow and fundamentally altering the bone marrow microenvironment.

Disclosures
No relevant conflicts of interest to declare.