Chronic myeloid leukemia (CML), driven by the fusion protein BCR-ABL1, remains highly responsive to treatment with tyrosine kinase inhibitors (TKIs). Although overt resistance and relapse typically occurs through on-target kinase domain mutations, 25-50% of all resistant cases lack such mutations. Second generation BCR-ABL1 inhibitors, such as dasatinib and nilotinib, retain the ability to inhibit a number of kinase domain mutations. Still a number of mutations, notably the T315I gatekeeper mutation, provides continued resistance and only recently with the approval of ponatinib are we able to inhibit all known kinase domain mutations in BCR-ABL1. With ponatinib approval effective inhibitors for all known drug-resistant mutations are available suggesting that poorly understood overt off-target resistance mechanisms should henceforth constitute a greater percentage of overt clinical resistance. Through the use of CML patient samples we sought to define molecular mediators of disease persistence (the occurrence of a hematologic but no cytogenetic response) and overt off-target resistance (a complete loss of cellular response to BCR-ABL1 inhibition), two poorly understood mechanisms of resistance in CML. Chronic phase CML patients exhibiting overexpression of the EVI1 oncogene displayed disease persistence, while the identification of two blast crisis CML patients exhibiting overt off-target resistance were found to contain EVI1 overexpression in conjunction with activated NRAS. Validation of NRAS Q61K, in CML cell lines, exhibited maintenance of cell viability despite TKI treatment and this overt resistance correlated with increased MAPK signaling. Furthermore, expression of MEK-DD, an activated MEK allele, provided a degree of resistance comparable that seen in NRAS Q61K driven off-target resistance. This establishes MAPK signaling as the main culprit downstream of NRAS that mediates the resistant phenotype. This was further substantiated by identifying sensitivity to MEK inhibition both alone and to an even greater extent in combination with BCR-ABL1 inhibitors. In all, we show that EVI1 overexpression can contribute to disease persistence and the presence of activating RAS mutations can lead to overt resistance to BCR-ABL1 tyrosine kinase inhibitors.

DNA damage is deleterious to all cells, but especially to stem cells because they maintain tissue integrity by constantly self-renewing (to replicate themselves) and differentiating (to produce all mature cells of the tissue). Hence, damage occurring in stem cells can be transmitted both horizontally (to other stem cells) and vertically (to differentiating cells). To study the DNA damage response (DDR) and DNA repair mechanisms employed by stem cells, we used hematopoietic stem cells (HSCs) isolated from young adult mice. We found that quiescent HSCs have cell-intrinsic mechanisms ensuring their survival in response to ionizing radiation (IR), including enhanced pro-survival gene expression and activation of p53-mediated DDR. We show how nonhomologous end joining (NHEJ) DNA repair in quiescent HSCs is associated with acquisition of genomic rearrangements, which can persist in vivo and contribute to hematopoietic abnormalities. Hence, we demonstrate that quiescence, long believed to be strictly protective, actually has a dark side and renders HSCs intrinsically vulnerable to mutagenesis. It has been suggested that the decline of tissue homeostasis seen with age may be caused by impaired stem cell activity. To understand how aging affects DNA repair capability of stem cells we studied the DDR and repair of HSCs isolated from old mice. We confirmed the previously documented numerical expansion, lineage skewing, and high basal levels of genomic stress in old HSCs. We found that old HSCs have a G1 arrest and exhibit a senescence phenotype compared to young HSCs. We show that the intrinsic genomic stress in old HSCs happens at the nucleolus and telomeres, two known fragile sites of the genome, and is potentially caused by replication stress. We demonstrate that old HSCs use NHEJ to repair IR induced DNA damage as efficiently as young HSCs. Taken together, these data indicate that old HSCs have decreased protective mechanisms and increased levels of intrinsic stress that may account for their decreased functionality. Moreover, they suggest that the life-long use of error-prone NHEJ repair in HSCs may be the driving force for the increased cancer incidence occurring with age in the hematopoietic system.

Aging is the greatest risk factor for a wide range of diseases, including cardiovascular diseases, cancers, neurodegenerative disorders, and diabetes. Yet why this is happening mechanistically remains largely unknown. Stem cells are responsible for the lifelong generation, maintenance, and repair of tissue and organ systems, and are therefore are both useful models to study cellular aging and attractive candidates for causing tissue and organismal aging. While stem cell aging has not been definitively linked to organismal aging, a decline in tissue function with age often correlates with a decline in stem cell function. Aging is also often accompanied by a decline in stem cell function, often correlating with degenerative diseases. In particular, during aging, hematopoietic stem cells (HSCs) lose their ability to regenerate the blood system, and promote disease development. Autophagy is critical for protecting HSCs from metabolic stress and has long been associated with health and longevity. Here, we show that loss of autophagy in HSCs causes accumulation of mitochondria and an activated metabolic state, which drives accelerated myeloid differentiation mainly through epigenetic deregulations, and impairs HSC self-renewal activity and regenerative potential. Strikingly, the majority of HSCs in aged mice share these altered metabolic and functional features. However, ~ 1/3 of aged HSCs exhibit high autophagy levels and maintain a low metabolic state with robust long-term regeneration potential similar to healthy young HSCs. Our results demonstrate that autophagy actively suppresses HSC metabolism by clearing active, healthy mitochondria to maintain quiescence and stemness, and becomes increasingly necessary with age to preserve the regenerative capacity of old HSCs.

The hematopoietic stem cell (HSC) and the granulocyte/macrophage progenitor (GMP), a downstream myeloid progenitor, reside within distinct bone marrow (BM) niches. The biophysical properties of these BM niches and the effect of these biophysical properties on the regulation of HSCs and GMP cell function remain unknown. We determined the mechanical properties of the endosteum, the perivasculature and non-vascularized central marrow, three specific BM niches that HSCs or GMPs have been shown to reside. We then used ex vivo adhesion assays to determine the extracellular matrix (ECM) ligands to which HSCs and GMPs preferentially adhere. Ex vivo culture on polyacrylamide (PA) hydrogels mimicking distinct BM niche microenvironments were then used to assay the effect of ECM elasticity on cell expansion and lineage differentiation capacity. We also performed transplantation assays after HSC expansion on PA gels to determine the role of ECM elasticity on engraftment potential. We found that GMPs preferentially bound to fibrillar collagen, but also adhered to fibronectin, and showed greater expansion on soft, compliant environments. Conversely, HSCs adhered only to fibronectin, and after growth on stiff environments, had greater cell expansion and higher engraftment potential due to increased expression of homing receptors. Myeloid differentiation was found to be affected by ECM elasticity such that compliant ECM niches enhanced granulocyte production. Conversely, stiff environments enhanced macrophage lineage commitment. Mechanotransduction of ECM elasticity was determined to be dependent on actomyosin contraction and activation of focal adhesion kinase (FAK) likely through integrin engagement. Together, our data show that the biophysical properties of specific BM niches function in controlling the differential growth and differentiation of HSCs and progenitors during homeostasis, and may even have the potential to regulate hematopoietic regeneration following insult, injury or transplantation.