John D. Crispino

DCAF7 is one of the few DCAFs expressed in hematopoietic stem cells (HSCs). Using a conditional knockout mouse model, we discovered that the absence of DCAF7 leads to accumulation of differentiating progenitors in the bone marrow, and impairs self-renewal capacity of HSCs upon transplantation. Furthermore, loss of Dcaf7 accelerates the differentiation of HSCs into the myeloid lineage. At the molecular level, DCAF7 interacts with components of the polycomb repressive complex 1 (PRC1) and promotes their assembly into large macromolecular complexes. Chromatin profiling revealed that loss of DCAF7 leads to increased localization of RING1B at transcriptionally active genomic loci. This increased binding was observed at genes involved in myeloid differentiation and was associated with increased mRNA expression. Lastly, inhibition of KDM2/7 proteins rescues the defective growth of Dcaf7 null HSCs in vitro. Together, these data reveal that DCAF7 is a novel regulator of the PRC1 complex and epigenetic enzymes that control HSC differentiation.

Children with Down syndrome (DS) frequently develop transient abnormal myelopoiesis that can evolve to the myeloid leukemia of DS (ML-DS). TAM spontaneously resolves in most cases but progresses to ML-DS with additional mutations, most commonly in the cohesin complex. However, the mechanisms by which these alterations promote leukemia are unknown. We leveraged the RAD21-mutant CMY cell line and RAD21-corrected CMY isogenic clones, as well as patient data, to investigate the effect of cohesin mutations during leukemia progression. RNA-sequencing revealed that HLA-class II genes were significantly down-regulated with cohesin mutations. Furthermore, HLA-DR was found to be lower in ML-DS relative to TAM, and these decreased levels were associated with increased risk of leukemia progression. Multi-omic analyses revealed that haploinsufficiency of RAD21 altered chromatin accessibility and impaired the occupancy of GATA1s and CIITA, the master regulator of HLA-class II gene expression. Chromatin binding of CIITA was increased with RAD21 correction, providing a mechanism by which restoration of cohesin improves HLA-class II expression. Finally, decreased levels of RAD21 or STAG2 expression in other subtypes of AML also exhibited reduced expression of HLA-class II genes. Thus, cohesin may contribute to leukemia by altering HLA-class II gene expression.

Mutations in GATA1 that cause skipping of exon 2, which encodes the N terminus, are associated with the myeloid leukemia of Down syndrome and Diamond-Blackfan anemia (DBA). To elucidate the molecular function of this N-terminal region, we used single-cell RNA sequencing (scRNA-seq) on fetal liver cells from Gata1-mutant embryos that express only the short isoform of GATA1 (GATA1s) lacking the N terminus of full-length GATA1 (GATA1FL). scRNA-seq revealed defects in erythropoiesis and aberrant upregulation of glycolytic genes, including PKM, which encodes pyruvate kinase to catalyze the final and irreversible step of glycolysis. Using precision nuclear run-on sequencing and cleavage under targets and release using nuclease (CUT&RUN) after acute GATA1 deletion in erythroid cells, we identified PKM as a direct target of GATA1. Substitution of GATA1FL with GATA1s induced histone lactylation at the PKM promoter, increased pyruvate kinase M (PKM) expression and activity, and enhanced glycolytic flux in erythroid progenitors, without affecting mitochondrial respiration. Importantly, PKM expression is also significantly elevated in patients with DBA with RPS19 mutations, which is associated with reduced levels of GATA1, further supporting a link between GATA1s-driven defective erythropoiesis and dysregulated glycolysis. Together, these findings reveal that GATA1 controls not only heme metabolism but also glycolytic reprogramming.

Diamond Blackfan anemia syndrome (DBAS) is a congenital ribosomopathy caused by haploinsufficiency of ribosomal proteins (RPs), but how RP stoichiometry and activity regulates erythroid development remains enigmatic. Using in vivo models, we uncover divergent functions for the small and large ribosomal subunit proteins RPS19 and RPL5 in fetal hematopoiesis. While RPL5 haploinsufficiency causes hematopoietic stem and progenitor cell (HSPC) accumulation and prenatal lethality via p53-mediated ferroptosis of mature erythroid progenitors, RPS19 haploinsufficiency leads to HSPC depletion and impaired erythroid expansion through p53-dependent apoptosis. The latter is accompanied by translational and transcriptional dysregulation, including the upregulation of RUNX1, which is also observed in RPS- haploinsufficient DBAS patients. Importantly, Runx1 deletion in RPS19-haploinsufficient mice partially rescues HSPC numbers. These findings reveal subunit-specific RP functions in controlling fetal hematopoiesis and demonstrate how imbalanced RP stoichiometry disrupts developmental programs, providing crucial mechanistic insights into DBAS pathogenesis and the basis for its clinical heterogeneity.

Myelodysplastic syndromes (MDSs) are malignant hematopoietic stem and progenitor cell (HSPC) disorders that lead to ineffective blood production with poor outcomes. We previously showed that F-box only protein 11 (FBXO11) is downregulated in MDS, and here we report how this event contributes to disease progression. Integration of multiomics data revealed that the SCF-FBXO11 complex regulates spliceosome and ribosome components in a nucleophosmin 1 (NPM1)-centric network. FBXO11 facilitates the ubiquitylation of NPM1, whereby deletion of FBXO11 results in the reorganization of NPM1 and a de-repression of alternative splicing. Label-free total quantitative proteomics demonstrated that the FBXO11-NPM1 interactome was markedly downregulated in cells from patients with CD34 ⁺ MDS. In addition, we discovered that MYC was evicted from the FBXO11 promoter by TLR2 activation, revealing that it was a MYC target gene and explaining why FBXO11 expression was decreased in MDS. In MDS mouse models, genetic ablation of Fbxo11 exacerbated neutropenia concomitant with a profound decrease in NPM1 protein levels. Finally, we discovered rare mutations in FBXO11 , which mapped to a previously unstudied functional intrinsically disordered region (IDR) in the N-terminus responsible for binding NPM1. These data support a model in which FBXO11 rewires RNA binding and ribosomal subnetworks through ubiquitylation of NPM1, ultimately restricting MDS progression.

Diamond Blackfan anemia syndrome (DBAS) is a congenital ribosomopathy caused by haploinsufficiency of ribosomal proteins (RPs), but how RP stoichiometry and activity regulates erythroid development remains enigmatic. Using novel in vivo models, we uncover strikingly divergent functions for the small and large ribosomal subunit proteins RPS19 and RPL5 in fetal hematopoiesis. While RPL5 haploinsufficiency causes hematopoietic stem and progenitor cell (HSPC) accumulation and prenatal lethality via p53-mediated ferroptosis of mature erythroid progenitors, RPS19 haploinsufficiency leads to HSPC depletion and impaired erythroid expansion through p53-dependent apoptosis. The latter is accompanied by translational and transcriptional dysregulation, including the upregulation of RUNX1 , which is also observed in RPS- haploinsufficient DBAS patients. Importantly, Runx1 deletion in RPS19-haploinsufficient mice partially rescues HSPC numbers. These findings reveal subunit-specific RP functions in controlling fetal hematopoiesis and demonstrate how imbalanced RP stoichiometry disrupts developmental programs, providing crucial mechanistic insights into DBAS pathogenesis and the basis for its clinical heterogeneity.

Chromothripsis, the chaotic shattering and repair of chromosomes, is common in cancer. Whether chromothripsis generates actionable therapeutic targets remains an open question. In a cohort of 64 patients in blast phase of a myeloproliferative neoplasm (BP-MPN), we describe recurrent amplification of a region of chromosome 21q (‘chr. 21amp’) in 25%, driven by chromothripsis in a third of these cases. We report that chr. 21amp BP-MPN has a particularly aggressive and treatment-resistant phenotype. DYRK1A, a serine threonine kinase, is the only gene in the 2.7-megabase minimally amplified region that showed both increased expression and chromatin accessibility compared with non-chr. 21amp BP-MPN controls. DYRK1A is a central node at the nexus of multiple cellular functions critical for BP-MPN development and is essential for BP-MPN cell proliferation in vitro and in vivo, and represents a druggable axis. Collectively, these findings define chr. 21amp as a prognostic biomarker in BP-MPN, and link chromothripsis to a therapeutic target.