Single-cell analyses reveal aberrant pathways for megakaryocyte-biased hematopoiesis in myelofibrosis and identify mutant clone-specific targets

Myelofibrosis is a severe myeloproliferative neoplasm characterized by increased numbers of abnormal bone marrow megakaryocytes that induce fibrosis, destroying the hematopoietic microenvironment. To determine the cellular and molecular basis for aberrant megakaryopoiesis in myelofibrosis, we performed single-cell transcriptome profiling of 135,929 CD34+ lineage− hematopoietic stem and progenitor cells (HSPCs), single-cell proteomics, genomics, and functional assays. We identified a bias toward megakaryocyte differentiation apparent from early multipotent stem cells in myelofibrosis and associated aberrant molecular signatures. A sub-fraction of myelofibrosis megakaryocyte progenitors (MkPs) are transcriptionally similar to healthy-donor MkPs, but the majority are disease specific, with distinct populations expressing fibrosis- and proliferation-associated genes. Mutant-clone HSPCs have increased expression of megakaryocyte-associated genes compared to wild-type HSPCs, and we provide early validation of G6B as a potential immunotherapy target. Our study paves the way for selective targeting of the myelofibrosis clone and illustrates the power of single-cell multi-omics to discover tumor-specific therapeutic targets and mediators of tissue fibrosis

Neck-motor interactions trigger rotation of the kinesin stalk

Rotation of the coiled-coil stalk of the kinesin-14 motors is thought to drive displacements or steps by the motor along microtubules, but the structural changes that trigger stalk rotation and the nucleotide state in which it occurs are not certain. Here we report a kinesin-14 neck mutant that releases ADP more slowly than wild type and shows weaker microtubule affinity, consistent with defective stalk rotation. Unexpectedly, crystal structures show the stalk fully rotated – neck-motor interactions destabilize the stalk, causing it to rotate and ADP to be released, and alter motor affinity for microtubules. A new structural pathway accounts for the coupling of stalk rotation – the force-producing stroke – to changes in motor affinity for nucleotide and microtubules. Sequential disruption of salt bridges that stabilize the unrotated stalk could cause the stalk to initiate and complete rotation in different nucleotide states

Transitions in lineage specification and gene regulatory networks in hematopoietic stem/progenitor cells over human development

Human hematopoiesis is a dynamic process that starts in utero 18–21 days post-conception. Understanding the site- and stage-specific variation in hematopoiesis is important if we are to understand the origin of hematological disorders, many of which occur at specific points in the human lifespan. To unravel how the hematopoietic stem/progenitor cell (HSPC) compartment changes during human ontogeny and the underlying gene regulatory mechanisms, we compare 57,489 HSPCs from 5 different tissues spanning 4 developmental stages through the human lifetime. Single-cell transcriptomic analysis identifies significant site- and developmental stage-specific transitions in cellular architecture and gene regulatory networks. Hematopoietic stem cells show progression from cycling to quiescence and increased inflammatory signaling during ontogeny. We demonstrate the utility of this dataset for understanding aberrant hematopoiesis through comparison to two cancers that present at distinct time points in postnatal life—juvenile myelomonocytic leukemia, a childhood cancer, and myelofibrosis, which classically presents in older adults

Single-cell analyses reveal megakaryocyte-biased hematopoiesis in myelofibrosis and identify mutant clone-specific targets

Myelofibrosis is a severe myeloproliferative neoplasm characterized by increased numbers of abnormal bone marrow megakaryocytes that induce fibrosis, destroying the hematopoietic microenvironment. To determine the cellular and molecular basis for aberrant megakaryopoiesis in myelofibrosis, we performed single-cell transcriptome profiling of 135,929 CD34+ lineage− hematopoietic stem and progenitor cells (HSPCs), single-cell proteomics, genomics, and functional assays. We identified a bias toward megakaryocyte differentiation apparent from early multipotent stem cells in myelofibrosis and associated aberrant molecular signatures. A sub-fraction of myelofibrosis megakaryocyte progenitors (MkPs) are transcriptionally similar to healthy-donor MkPs, but the majority are disease specific, with distinct populations expressing fibrosis- and proliferation-associated genes. Mutant-clone HSPCs have increased expression of megakaryocyte-associated genes compared to wild-type HSPCs, and we provide early validation of G6B as a potential immunotherapy target. Our study paves the way for selective targeting of the myelofibrosis clone and illustrates the power of single-cell multi-omics to discover tumor-specific therapeutic targets and mediators of tissue fibrosis

Single-cell profiling of human bone marrow progenitors reveals mechanisms of failing erythropoiesis in Diamond-Blackfan anemia

Ribosome dysfunction underlies the pathogenesis of many cancers and heritable ribosomopathies. Here, we investigate how mutations in either ribosomal protein large (RPL) or ribosomal protein small (RPS) subunit genes selectively affect erythroid progenitor development and clinical phenotypes in Diamond-Blackfan anemia (DBA), a rare ribosomopathy with limited therapeutic options. Using single-cell assays of patient-derived bone marrow, we delineated two distinct cellular trajectories segregating with ribosomal protein genotypes. Almost complete loss of erythroid specification was observed in RPS-DBA. In contrast, we observed relative preservation of qualitatively abnormal erythroid progenitors and precursors in RPL-DBA. Although both DBA genotypes exhibited a proinflammatory bone marrow milieu, RPS-DBA was characterized by erythroid differentiation arrest, whereas RPL-DBA was characterized by preserved GATA1 expression and activity. Compensatory stress erythropoiesis in RPL-DBA exhibited disordered differentiation underpinned by an altered glucocorticoid molecular signature, including reduced ZFP36L2 expression, leading to milder anemia and improved corticosteroid response. This integrative analysis approach identified distinct pathways of erythroid failure and defined genotype-phenotype correlations in DBA. These findings may help facilitate therapeutic target discovery