Autophagy in Epidermis.

PhDOrgan‐transplant recipients (OTRs) on a new class of immunosuppressants, rapamycin and its analogues, have reduced cutaneous Squamous Cell Carcinomas (cSCCs). Rapamycin, an mTORC1 inhibitor, is also a known autophagy inducer in experimental models. Autophagy, which literally means self‐eating, is a cell survival mechanism but can also lead to cell death. Therefore, the main hypothesis behind this work is that rapamycin prevents epidermal tumourigenesis by either affecting epidermal mTOR regulation of autophagy and/or selectively affecting epidermal AKT isoform activity. Epidermal keratinocytes move from the proliferating basal layer upwards to the granular layers where they terminally differentiate, forming a layer of flattened, anucleate cells or squames of the cornified layer which provides an essential environmental barrier. However, epidermal terminal differentiation, a specialised form of cell death involving organelle degradation, is poorly understood. The work presented in this thesis shows that analysis of the autophagy marker expression profile during foetal epidermal development, indicates autophagy is constitutively active in the terminally differentiating granular layer of epidermis. Therefore, I hypothesize that autophagy is a mechanism of organelle degradation during terminal differentiation of granular layer keratinocytes. In monolayer keratinocytes, activation of terminal differentiation is accompanied by autophagic degradation of nuclear material, nucleophagy. This suggests that constitutive autophagy is a pro‐death mechanism required for terminal differentiation. In cultured keratinocytes and in epidermal cultures, rapamycinmediated mTORC1 inhibition strongly increases AKT1 activity as well as up‐regulates constitutive granular layer autophagy promoting terminal differentiation. Therefore, autophagy is an important fundamental process in keratinocytes which may be the mechanism by which terminally differentiating keratinocytes of the epidermal granular layer degrade their organelles required for barrier formation. This may have implications for the treatment of patients with barrier defects like psoriasis. In immunosuppressed OTRs, rapamycin may promote epidermal autophagy and AKT1 activity adding to its anti‐tumourigenic properties

iASPP is a novel autophagy inhibitor in keratinocytes

The protein iASPP (encoded by PPP1R13L) is an evolutionarily conserved p53 inhibitor, the expression of which is often upregulated in human cancers. We have recently shown that iASPP is a crucial regulator of epidermal homeostasis. Here, we report that iASPP also acts as autophagy inhibitor in keratinocytes. Our data show that depletion of iASPP protects keratinocytes from apoptosis by modulating the expression of Noxa (also known as PMAIP1). In our model, iASPP expression can affect the fission-fusion cycle, mass and shape of mitochondria. iASPP-silenced keratinocytes display disorganization of cytosolic compartments and increased metabolic stress caused by deregulation of mTORC1 signaling. Moreover, increased levels of lipidated LC3 protein confirmed the activation of autophagy in iASPP-depleted cells. We have identified a novel mechanism modulating autophagy in keratinocytes that relies upon iASPP expression specifically reducing the interaction of Atg5-Atg12 with Atg16L1, an interaction that is essential for autophagosome formation or maturation. Using organotypic culture, we further explored the link between autophagy and differentiation, and we showed that impairing autophagy affects epidermal terminal differentiation. Our data provide an alternative mechanism to explain how epithelial integrity is maintained against environmental stressors and might also improve the understanding of the etiology of skin diseases that are characterized by defects in differentiation and DNA damage responses.15 page(s

Proliferation dynamics of acute myeloid leukaemia and haematopoietic progenitors competing for bone marrow space

Leukaemia progressively invades bone marrow (BM), outcompeting healthy haematopoiesis by mechanisms that are not fully understood. Combining cell number measurements with a short-timescale dual pulse labelling method, we simultaneously determine the proliferation dynamics of primitive haematopoietic compartments and acute myeloid leukaemia (AML). We observe an unchanging proportion of AML cells entering S phase per hour throughout disease progression, with substantial BM egress at high levels of infiltration. For healthy haematopoiesis, we find haematopoietic stem cells (HSCs) make a significant contribution to cell production, but we phenotypically identify a quiescent subpopulation with enhanced engraftment ability. During AML progression, we observe that multipotent progenitors maintain a constant proportion entering S phase per hour, despite a dramatic decrease in the overall population size. Primitive populations are lost from BM with kinetics that are consistent with ousting irrespective of cell cycle state, with the exception of the quiescent HSC subpopulation, which is more resistant to elimination

Constitutive Autophagy and Nucleophagy during Epidermal Differentiation

Funding support was provided by MRC, Cancer Research-UK and Barts, and The London Charity

Inhibition of Endosteal Vascular Niche Remodeling Rescues Hematopoietic Stem Cell Loss in AML

Bone marrow vascular niches sustain hematopoietic stem cells (HSCs) and are drastically remodeled in leukemia to support pathological functions. Acute myeloid leukemia (AML) cells produce angiogenic factors, which likely contribute to this remodeling, but anti-angiogenic therapies do not improve AML patient outcomes. Using intravital microscopy, we found that AML progression leads to differential remodeling of vasculature in central and endosteal bone marrow regions. Endosteal AML cells produce pro-inflammatory and anti-angiogenic cytokines and gradually degrade endosteal endothelium, stromal cells, and osteoblastic cells, whereas central marrow remains vascularized and splenic vascular niches expand. Remodeled endosteal regions have reduced capacity to support non-leukemic HSCs, correlating with loss of normal hematopoiesis. Preserving endosteal endothelium with the small molecule deferoxamine or a genetic approach rescues HSCs loss, promotes chemotherapeutic efficacy, and enhances survival. These findings suggest that preventing degradation of the endosteal vasculature may improve current paradigms for treating AML

T-cell acute leukaemia exhibits dynamic interactions with bone marrow microenvironments

It is widely accepted that complex interactions between cancer cells and their surrounding microenvironment contribute to disease development, chemo-resistance and disease relapse. In light of this observed interdependency, novel therapeutic interventions that target specific cancer stroma cell lineages and their interactions are being sought. Here we studied a mouse model of human T-cell acute lymphoblastic leukaemia (T-ALL) and used intravital microscopy to monitor the progression of disease within the bone marrow at both the tissue-wide and single-cell level over time, from bone marrow seeding to development/selection of chemo-resistance. We observed highly dynamic cellular interactions and promiscuous distribution of leukaemia cells that migrated across the bone marrow, without showing any preferential association with bone marrow sub-compartments. Unexpectedly, this behaviour was maintained throughout disease development, from the earliest bone marrow seeding to response and resistance to chemotherapy. Our results reveal that T-ALL cells do not depend on specific bone marrow microenvironments for propagation of disease, nor for the selection of chemo-resistant clones, suggesting that a stochastic mechanism underlies these processes. Yet, although T-ALL infiltration and progression are independent of the stroma, accumulated disease burden leads to rapid, selective remodelling of the endosteal space, resulting in a complete loss of mature osteoblastic cells while perivascular cells are maintained. This outcome leads to a shift in the balance of endogenous bone marrow stroma, towards a composition associated with less efficient haematopoietic stem cell function. This novel, dynamic analysis of T-ALL interactions with the bone marrow microenvironment in vivo, supported by evidence from human T-ALL samples, highlights that future therapeutic interventions should target the migration and promiscuous interactions of cancer cells with the surrounding microenvironment, rather than specific bone marrow stroma, to combat the invasion by and survival of chemo-resistant T-ALL cells