Reprogramming identifies functionally distinct stages of clonal evolution in myelodysplastic syndromes.

Myeloid neoplasms, including myelodysplastic syndromes (MDS), are genetically heterogeneous disorders driven by clonal acquisition of somatic mutations in hematopoietic stem and progenitor cells (HPCs). The order of premalignant mutations and their impact on HPC self-renewal and differentiation remain poorly understood. We show that episomal reprogramming of MDS patient samples generates induced pluripotent stem cells from single premalignant cells with a partial complement of mutations, directly informing the temporal order of mutations in the individual patient. Reprogramming preferentially captured early subclones with fewer mutations, which were rare among single patient cells. To evaluate the functional impact of clonal evolution in individual patients, we differentiated isogenic MDS induced pluripotent stem cells harboring up to 4 successive clonal abnormalities recapitulating a progressive decrease in hematopoietic differentiation potential. SF3B1, in concert with epigenetic mutations, perturbed mitochondrial function leading to accumulation of damaged mitochondria during disease progression, resulting in apoptosis and ineffective erythropoiesis. Reprogramming also informed the order of premalignant mutations in patients with complex karyotype and identified 5q deletion as an early cytogenetic anomaly. The loss of chromosome 5q cooperated with TP53 mutations to perturb genome stability, promoting acquisition of structural and karyotypic abnormalities. Reprogramming thus enables molecular and functional interrogation of preleukemic clonal evolution, identifying mitochondrial function and chromosome stability as key pathways affected by acquisition of somatic mutations in MDS.

Adult Expression of Tbr2 Is Required for the Maintenance but Not Survival of Intrinsically Photosensitive Retinal Ganglion Cells.

Retinal ganglion cells expressing the photopigment melanopsin are intrinsically photosensitive (ipRGCs). ipRGCs regulate subconscious non-image-forming behaviors such as circadian rhythms, pupil dilation, and light-mediated mood. Previously, we and others showed that the transcription factor Tbr2 (EOMES) is required during retinal development for the formation of ipRGCs. Tbr2 is also expressed in the adult retina leading to the hypothesis that it plays a role in adult ipRGC function. To test this, we removed Tbr2 in adult mice. We found that this results in the loss of melanopsin expression in ipRGCs but does not lead to cell death or morphological changes to their dendritic or axonal termination patterns. Additionally, we found ectopic expression of Tbr2 in conventional RGCs does not induce melanopsin expression but can increase melanopsin expression in existing ipRGCs. An interesting feature of ipRGCs is their superior survival relative to conventional RGCs after an optic nerve injury. We find that loss of Tbr2 decreases the survival rate of ipRGCs after optic nerve damage suggesting that Tbr2 plays a role in ipRGC survival after injury. Lastly, we show that the GABAergic amacrine cell marker Meis2, is expressed in the majority of Tbr2-expressing displaced amacrine cells as well as in a subset of Tbr2-expressing RGCs. These findings demonstrate that Tbr2 is necessary but not sufficient for melanopsin expression, that Tbr2 is involved in ipRGC survival after optic nerve injury, and identify a marker for Tbr2-expressing displaced amacrine cells.

ATG4A regulates human erythroid maturation and mitochondrial clearance.

Autophagy is a self-degradation pathway that is essential for erythropoiesis. During erythroid differentiation, autophagy facilitates the degradation of macromolecules and the programmed clearance of mitochondria. Impaired mitochondrial clearance results in anemia and alters the lifespan of red blood cells in vivo. While several essential autophagy genes contribute to autophagy in erythropoiesis, little is known about erythroid-specific mediators of this pathway. Genetic analysis of primary human erythroid and nonerythroid cells revealed the selective upregulation of the core autophagy gene ATG4A in maturing human erythroid cells. Because the function of ATG4A in erythropoiesis is unknown, we evaluated its role using an ex vivo model of human erythropoiesis. Depletion of ATG4A in primary human hematopoietic stem and progenitor cells selectively impaired erythroid but not myeloid lineage differentiation, resulting in reduced red cell production, delayed terminal differentiation, and impaired enucleation. Loss of ATG4A impaired autophagy and mitochondrial clearance, giving rise to reticulocytes with retained mitochondria and autophagic vesicles. In summary, our study identifies ATG4A as a cell type-specific regulator of autophagy in erythroid development.

Lamin B1 deletion in myeloid neoplasms causes nuclear anomaly and altered hematopoietic stem cell function.

Abnormal nuclear morphology is a hallmark of malignant cells widely used in cancer diagnosis. Pelger-Huët anomaly (PHA) is a common abnormality of neutrophil nuclear morphology of unknown molecular etiology in myeloid neoplasms (MNs). We show that loss of nuclear lamin B1 (LMNB1) encoded on chromosome 5q, which is frequently deleted in MNs, induces defects in nuclear morphology and human hematopoietic stem cell (HSC) function associated with malignancy. LMNB1 deficiency alters genome organization inducing in vitro and in vivo expansion of HSCs, myeloid-biased differentiation with impaired lymphoid commitment, and genome instability due to defective DNA damage repair. Nuclear dysmorphology of neutrophils in patients with MNs is associated with 5q deletions spanning the LMNB1 locus, and lamin B1 loss is both necessary and sufficient to cause PHA in normal and 5q-deleted neutrophils. LMNB1 loss thus causes acquired PHA and links abnormal nuclear morphology with HSCs and progenitor cell fate determination via genome organization.

Induced pluripotent stem cell models of myeloid malignancies and clonal evolution.

Reprogramming of cells from patients with genetic disorders to pluripotency is a promising avenue to understanding disease biology. A number of induced pluripotent stem cell (iPSC) models of inherited monogenic blood disorders have been reported over the past decade. However, the application of iPSCs for modeling of hematological malignancies has only recently been explored. Blood malignancies comprise a spectrum of genetically heterogeneous disorders marked by the acquisition of somatic mutations and chromosomal aberrations. This genetic heterogeneity presents unique challenges for iPSC modeling, but also opportunities to capture genetically distinct states and generate models of stepwise progression from normal to malignant hematopoiesis. Here we briefly review the current state of this field, highlighting current models of acquired pre-malignant and malignant blood disorders and clonal evolution, and challenges including barriers to reprogramming and differentiation of iPSCs into bona fide hematopoietic stem cells.