IL-27 maintains cytotoxic Ly6C+ γδ T cells that arise from immature precursors.

In mice, γδ-T lymphocytes that express the co-stimulatory molecule, CD27, are committed to the IFNγ-producing lineage during thymic development. In the periphery, these cells play a critical role in host defense and anti-tumor immunity. Unlike αß-T cells that rely on MHC-presented peptides to drive their terminal differentiation, it is unclear whether MHC-unrestricted γδ-T cells undergo further functional maturation after exiting the thymus. Here, we provide evidence of phenotypic and functional diversity within peripheral IFNγ-producing γδ T cells. We found that CD27+ Ly6C- cells convert into CD27+Ly6C+ cells, and these CD27+Ly6C+ cells control cancer progression in mice, while the CD27+Ly6C- cells cannot. The gene signatures of these two subsets were highly analogous to human immature and mature γδ-T cells, indicative of conservation across species. We show that IL-27 supports the cytotoxic phenotype and function of mouse CD27+Ly6C+ cells and human Vδ2+ cells, while IL-27 is dispensable for mouse CD27+Ly6C- cell and human Vδ1+ cell functions. These data reveal increased complexity within IFNγ-producing γδ-T cells, comprising immature and terminally differentiated subsets, that offer new insights into unconventional T-cell biology.

CML cells actively evade host immune surveillance through cytokine-mediated downregulation of MHC-II expression.

Targeting the fusion oncoprotein BCR-ABL with tyrosine kinase inhibitors has significantly affected chronic myeloid leukemia (CML) treatment, transforming the life expectancy of patients; however the risk for relapse remains, due to persistence of leukemic stem cells (LSCs). Therefore it is imperative to explore the mechanisms that result in LSC survival and develop new therapeutic approaches. We now show that major histocompatibility complex (MHC)-II and its master regulator class II transactivator (CIITA) are downregulated in CML compared with non-CML stem/progenitor cells in a BCR-ABL kinase-independent manner. Interferon γ (IFN-γ) stimulation resulted in an upregulation of CIITA and MHC-II in CML stem/progenitor cells; however, the extent of IFN-γ-induced MHC-II upregulation was significantly lower than when compared with non-CML CD34+ cells. Interestingly, the expression levels of CIITA and MHC-II significantly increased when CML stem/progenitor cells were treated with the JAK1/2 inhibitor ruxolitinib (RUX). Moreover, mixed lymphocyte reactions revealed that exposure of CD34+ CML cells to IFN-γ or RUX significantly enhanced proliferation of the responder CD4+CD69+ T cells. Taken together, these data suggest that cytokine-driven JAK-mediated signals, provided by CML cells and/or the microenvironment, antagonize MHC-II expression, highlighting the potential for developing novel immunomodulatory-based therapies to enable host-mediated immunity to assist in the detection and eradication of CML stem/progenitor cells.

The role of mTOR-mediated signals during haemopoiesis and lineage commitment.

The serine/threonine protein kinase mechanistic target of rapamycin (mTOR) has been implicated in the regulation of an array of cellular functions including protein and lipid synthesis, proliferation, cell size and survival. Here, we describe the role of mTOR during haemopoiesis within the context of mTORC1 and mTORC2, the distinct complexes in which it functions. The use of conditional transgenic mouse models specifically targeting individual mTOR signalling components, together with selective inhibitors, have generated a significant body of research emphasising the critical roles played by mTOR, and individual mTOR complexes, in haemopoietic lineage commitment and development. This review will describe the profound role of mTOR in embryogenesis and haemopoiesis, underscoring the importance of mTORC1 at the early stages of haemopoietic cell development, through modulation of stem cell potentiation and self-renewal, and erythroid and B cell lineage commitment. Furthermore, the relatively discrete role of mTORC2 in haemopoiesis will be explored during T cell development and B cell maturation. Collectively, this review aims to highlight the functional diversity of mTOR signalling and underline the importance of this pathway in haemopoiesis.

CXCR2 and CXCL4 regulate survival and self-renewal of hematopoietic stem/progenitor cells.

The regulation of hematopoietic stem cell (HSC) survival and self-renewal within the bone marrow (BM) niche is not well understood. We therefore investigated global transcriptomic profiling of normal human HSC/hematopoietic progenitor cells [HPCs], revealing that several chemokine ligands (CXCL1-4, CXCL6, CXCL10, CXCL11, and CXCL13) were upregulated in human quiescent CD34(+)Hoescht(-)Pyronin Y(-) and primitive CD34(+)38(-), as compared with proliferating CD34(+)Hoechst(+)Pyronin Y(+) and CD34(+)38(+) stem/progenitor cells. This suggested that chemokines might play an important role in the homeostasis of HSCs. In human CD34(+) hematopoietic cells, knockdown of CXCL4 or pharmacologic inhibition of the chemokine receptor CXCR2, significantly decreased cell viability and colony forming cell (CFC) potential. Studies on Cxcr2(-/-) mice demonstrated enhanced BM and spleen cellularity, with significantly increased numbers of HSCs, hematopoietic progenitor cell-1 (HPC-1), HPC-2, and Lin(-)Sca-1(+)c-Kit(+) subpopulations. Cxcr2(-/-) stem/progenitor cells showed reduced self-renewal capacity as measured in serial transplantation assays. Parallel studies on Cxcl4 demonstrated reduced numbers of CFC in primary and secondary assays following knockdown in murine c-Kit(+) cells, and Cxcl4(-/-) mice showed a decrease in HSC and reduced self-renewal capacity after secondary transplantation. These data demonstrate that the CXCR2 network and CXCL4 play a role in the maintenance of normal HSC/HPC cell fates, including survival and self-renewal.

Chromatin looping defines expression of TAL1, its flanking genes, and regulation in T-ALL.

TAL1 is an important regulator of hematopoiesis and its expression is tightly controlled despite complexities in its genomic organization. It is frequently misregulated in T-cell acute lymphoblastic leukemia (T-ALL), often due to deletions between TAL1 and the neighboring STIL gene. To better understand the events that lead to TAL1 expression in hematopoiesis and in T-ALL, we studied looping interactions at the TAL1 locus. In TAL1-expressing erythroid cells, the locus adopts a looping "hub" which brings into close physical proximity all known TAL1 cis-regulatory elements including CTCF-bound insulators. Loss of GATA1 results in disassembly of the hub and loss of CTCF/RAD21 from one of its insulators. Genes flanking TAL1 are partly dependent on hub integrity for their transcriptional regulation. We identified looping patterns unique to TAL1-expressing T-ALL cells, and, intriguingly, loops occurring between the TAL1 and STIL genes at the common TAL1/STIL breakpoints found in T-ALL. These findings redefine how TAL1 and neighboring genes communicate within the nucleus, and indicate that looping facilitates both normal and aberrant TAL1 expression and may predispose to structural rearrangements in T-ALL. We also propose that GATA1-dependent looping mechanisms may facilitate the conservation of TAL1 regulation despite cis-regulatory remodeling during vertebrate evolution.

Megakaryocytes assemble podosomes that degrade matrix and protrude through basement membrane.

Megakaryocytes give rise to platelets via extension of proplatelet arms, which are released through the vascular sinusoids into the bloodstream. Megakaryocytes and their precursors undergo varying interactions with the extracellular environment in the bone marrow during their maturation and positioning in the vascular niche. We demonstrate that podosomes are abundant in primary murine megakaryocytes adherent on multiple extracellular matrix substrates, including native basement membrane. Megakaryocyte podosome lifetime and density, but not podosome size, are dependent on the type of matrix, with podosome lifetime dramatically increased on collagen fibers compared with fibrinogen. Podosome stability and dynamics depend on actin cytoskeletal dynamics but not matrix metalloproteases. However, podosomes degrade matrix and appear to be important for megakaryocytes to extend protrusions across a native basement membrane. We thus demonstrate for the first time a fundamental requirement for podosomes in megakaryocyte process extension across a basement membrane, and our results suggest that podosomes may have a role in proplatelet arm extension or penetration of basement membrane.

Generation of a poor prognostic chronic lymphocytic leukemia-like disease model: PKCα subversion induces up-regulation of PKCßII expression in B lymphocytes.

Overwhelming evidence identifies the microenvironment as a critical factor in the development and progression of chronic lymphocytic leukemia, underlining the importance of developing suitable translational models to study the pathogenesis of the disease. We previously established that stable expression of kinase dead protein kinase C alpha in hematopoietic progenitor cells resulted in the development of a chronic lymphocytic leukemia-like disease in mice. Here we demonstrate that this chronic lymphocytic leukemia model resembles the more aggressive subset of chronic lymphocytic leukemia, expressing predominantly unmutated immunoglobulin heavy chain genes, with upregulated tyrosine kinase ZAP-70 expression and elevated ERK-MAPK-mTor signaling, resulting in enhanced proliferation and increased tumor load in lymphoid organs. Reduced function of PKCα leads to an up-regulation of PKCßII expression, which is also associated with a poor prognostic subset of human chronic lymphocytic leukemia samples. Treatment of chronic lymphocytic leukemia-like cells with the selective PKCß inhibitor enzastaurin caused cell cycle arrest and apoptosis both in vitro and in vivo, and a reduction in the leukemic burden in vivo. These results demonstrate the importance of PKCßII in chronic lymphocytic leukemia-like disease progression and suggest a role for PKCα subversion in creating permissive conditions for leukemogenesis.

Protein kinase C in cellular transformation: a valid target for therapy?

The protein kinase C (PKC) family of serine/threonine protein kinases share structural homology, while exhibiting substantial functional diversity. PKC isoforms are ubiquitously expressed in tissues which makes it difficult to define roles for individual isoforms, with complexity compounded by the finding that PKC isoforms can co-operate with or antagonize other PKC family members. A number of studies suggest the involvement of PKC family members in regulating leukaemic cell survival and proliferation. Chronic lymphocytic leukaemia (CLL), the most common leukaemia in the Western world, exhibits dysregulated expression of PKC isoforms, with recent reports indicating that PKCß and δ play a critical role in B-cell development, due to their ability to link the B-cell receptor (BCR) with downstream signalling pathways. Given the prognostic significance of the BCR in CLL, inhibition of these BCR/PKC-mediated signalling pathways is of therapeutic relevance. The present review discusses the emerging role of PKC isoforms in the pathophysiology of CLL and assesses approaches that have been undertaken to modulate PKC activity.

The MYC-NFATC2 axis maintains the cell cycle and mitochondrial function in acute myeloid leukaemia cells.

Acute myeloid leukaemia (AML) is a clonal haematological malignancy affecting the myeloid lineage, with generally poor patient outcomes owing to the lack of targeted therapies. The histone lysine demethylase 4A (KDM4A) has been established as a novel therapeutic target in AML, due to its selective oncogenic role within leukaemic cells. We identify that the transcription factor nuclear factor of activated T cells 2 (NFATC2) is a novel binding and transcriptional target of KDM4A in the human AML THP-1 cell line. Furthermore, cytogenetically diverse AML cell lines, including THP-1, were dependent on NFATC2 for colony formation in vitro, highlighting a putative novel mechanism of AML oncogenesis. Our study demonstrates that NFATC2 maintenance of cell cycle progression in human AML cells was driven primarily by CCND1. Through RNA sequencing (RNA-seq) and chromatin immunoprecipitation sequencing (ChIP-seq), NFATc2 was shown to bind to the promoter region of genes involved in oxidative phosphorylation and subsequently regulate their gene expression in THP-1 cells. Furthermore, our data show that NFATC2 shares transcriptional targets with the transcription factor c-MYC, with MYC knockdown phenocopying NFATC2 knockdown. These data suggest a newly identified co-ordinated role for NFATC2 and MYC in the maintenance of THP-1 cell function, indicative of a potential means of therapeutic targeting in human AML.

Activating p53 abolishes self-renewal of quiescent leukaemic stem cells in residual CML disease.

Whilst it is recognised that targeting self-renewal is an effective way to functionally impair the quiescent leukaemic stem cells (LSC) that persist as residual disease in chronic myeloid leukaemia (CML), developing therapeutic strategies to achieve this have proved challenging. We demonstrate that the regulatory programmes of quiescent LSC in chronic phase CML are similar to that of embryonic stem cells, pointing to a role for wild type p53 in LSC self-renewal. In support of this, increasing p53 activity in primitive CML cells using an MDM2 inhibitor in combination with a tyrosine kinase inhibitor resulted in reduced CFC outputs and engraftment potential, followed by loss of multilineage priming potential and LSC exhaustion when combination treatment was discontinued. Our work provides evidence that targeting LSC self-renewal is exploitable in the clinic to irreversibly impair quiescent LSC function in CML residual disease - with the potential to enable more CML patients to discontinue therapy and remain in therapy-free remission.

Modulation of PKC-α promotes lineage reprogramming of committed B lymphocytes.

During hematopoietic lineage development, hematopoietic stem cells sequentially commit toward myeloid or lymphoid lineages in a tightly regulated manner, which under normal circumstances is irreversible. However, studies have established that targeted deletion of the B-lineage specific transcription factor, paired box gene 5 (Pax5), enables B cells to differentiate toward other hematopoietic lineages, in addition to generating progenitor B-cell lymphomas. Our previous studies showed that subversion of protein kinase C (PKC)-α in developing B cells transformed B-lineage cells. Here, we demonstrate that PKC-α modulation in committed CD19(+) B lymphocytes also promoted lineage conversion toward myeloid, NK-, and T-cell lineages upon Notch ligation. This occurred via a reduction in Pax5 expression resulting from a downregulation of E47, a product of the E2A gene. T-cell lineage commitment was indicated by the expression of T-cell associated genes Ptcra, Cd3e, and gene rearrangement at the Tcrb gene locus. Importantly, the lineage-converted T cells carried Igh gene rearrangements reminiscent of their B-cell origin. Our findings suggest that modulation of PKC-α induces hematopoietic-lineage plasticity in committed B-lineage cells by perturbing expression of critical B-lineage transcription factors, and deregulation of PKC-α activity/expression represents a potential mechanism for lineage trans-differentiation during malignancies.

The discrete roles of individual FOXO transcription factor family members in B-cell malignancies.

Forkhead box (FOX) class O (FOXO) proteins are a dynamic family of transcription factors composed of four family members: FOXO1, FOXO3, FOXO4 and FOXO6. As context-dependent transcriptional activators and repressors, the FOXO family regulates diverse cellular processes including cell cycle arrest, apoptosis, metabolism, longevity and cell fate determination. A central pathway responsible for negative regulation of FOXO activity is the phosphatidylinositol-3-kinase (PI3K)-AKT signalling pathway, enabling cell survival and proliferation. FOXO family members can be further regulated by distinct kinases, both positively (e.g., JNK, AMPK) and negatively (e.g., ERK-MAPK, CDK2), with additional post-translational modifications further impacting on FOXO activity. Evidence has suggested that FOXOs behave as 'bona fide' tumour suppressors, through transcriptional programmes regulating several cellular behaviours including cell cycle arrest and apoptosis. However, an alternative paradigm has emerged which indicates that FOXOs operate as mediators of cellular homeostasis and/or resistance in both 'normal' and pathophysiological scenarios. Distinct FOXO family members fulfil discrete roles during normal B cell maturation and function, and it is now clear that FOXOs are aberrantly expressed and mutated in discrete B-cell malignancies. While active FOXO function is generally associated with disease suppression in chronic lymphocytic leukemia for example, FOXO expression is associated with disease progression in diffuse large B cell lymphoma, an observation also seen in other cancers. The opposing functions of the FOXO family drives the debate about the circumstances in which FOXOs favour or hinder disease progression, and whether targeting FOXO-mediated processes would be effective in the treatment of B-cell malignancies. Here, we discuss the disparate roles of FOXO family members in B lineage cells, the regulatory events that influence FOXO function focusing mainly on post-translational modifications, and consider the potential for future development of therapies that target FOXO activity.

mTORC1-selective activation of translation elongation promotes disease progression in chronic lymphocytic leukemia.

Targeted deletion of Raptor, a component of mechanistic target of rapamycin complex 1 (mTORC1), reveals an essential role for mTORC1 in initiation/maintenance of leukemia in a CLL model, resulting from a failure for haemopoietic stem/progenitor cells (HSPCs) to commit to the B cell lineage. Induction of Raptor-deficiency in NSG mice transplanted with Mx1-Raptor CLL progenitor cells (PKCα-KR-transduced HSPCs) after disease establishment revealed a reduction in CLL-like disease load and a significant increase in survival in the mice. Interestingly in an aggressive CLL-like disease model, rapamycin treatment reduced disease burden more effectively than AZD2014 (dual mTORC1/2 inhibitor), indicating a skew towards mTORC1 sensitivity with more aggressive disease. Rapamycin, but not ibrutinib, efficiently targeted the eEF2/eEF2K translation elongation regulatory axis, downstream of mTORC1, resulting in eEF2 inactivation through induction of eEF2T56 phosphorylation. mTOR inhibitor treatment of primary patient CLL cells halted proliferation, at least in part through modulation of eEF2K/eEF2 phosphorylation and expression, reduced protein synthesis and inhibited expression of MCL1, Cyclin A and Cyclin D2. Our studies highlight the importance of translation elongation as a driver of disease progression and identify inactivation of eEF2 activity as a novel therapeutic target for blocking CLL progression.

Pyruvate anaplerosis is a targetable vulnerability in persistent leukaemic stem cells.

Deregulated oxidative metabolism is a hallmark of leukaemia. While tyrosine kinase inhibitors (TKIs) such as imatinib have increased survival of chronic myeloid leukaemia (CML) patients, they fail to eradicate disease-initiating leukemic stem cells (LSCs). Whether TKI-treated CML LSCs remain metabolically deregulated is unknown. Using clinically and physiologically relevant assays, we generate multi-omics datasets that offer unique insight into metabolic adaptation and nutrient fate in patient-derived CML LSCs. We demonstrate that LSCs have increased pyruvate anaplerosis, mediated by increased mitochondrial pyruvate carrier 1/2 (MPC1/2) levels and pyruvate carboxylase (PC) activity, in comparison to normal counterparts. While imatinib reverses BCR::ABL1-mediated LSC metabolic reprogramming, stable isotope-assisted metabolomics reveals that deregulated pyruvate anaplerosis is not affected by imatinib. Encouragingly, genetic ablation of pyruvate anaplerosis sensitises CML cells to imatinib. Finally, we demonstrate that MSDC-0160, a clinical orally-available MPC1/2 inhibitor, inhibits pyruvate anaplerosis and targets imatinib-resistant CML LSCs in robust pre-clinical CML models. Collectively these results highlight pyruvate anaplerosis as a persistent and therapeutically targetable vulnerability in imatinib-treated CML patient-derived samples.

Immune complex-mediated co-ligation of the BCR with FcγRIIB results in homeostatic apoptosis of B cells involving Fas signalling that is defective in the MRL/Lpr model of systemic lupus erythematosus.

Negative regulation of B cell activation by cognate immune complexes plays an important homeostatic role in suppressing B cell hyperactivity and preventing consequent autoimmunity. Immune complexes co-ligate the BCR and FcγRIIB resulting in both growth arrest and apoptosis. We now show that such apoptotic signalling involves induction and activation of p53 and its target genes, the pro-apoptotic Bcl-2 family members, Bad and Bid, as well as nuclear export of p53. Collectively, these events result in destabilisation of the mitochondrial and lysosomal compartments with consequent activation and interplay of executioner caspases and endosomal-derived proteases. In addition, the upregulation of Fas and FasL with consequent activation of caspase 8-dependent death receptor signalling is required to facilitate efficient apoptosis of B cells. Consistent with this role for Fas death receptor signalling, apoptosis resulting from co-ligation of the BCR and FcγRIIB is defective in B cells from Fas-deficient MRL/MpJ-Fas(lpr) mice. As these mice develop spontaneous, immune complex-driven lupus-like glomerulonephritis, targeting this FcγRIIB-mediated apoptotic pathway may therefore have novel therapeutic implications for systemic autoimmune disease.

PKCß Facilitates Leukemogenesis in Chronic Lymphocytic Leukaemia by Promoting Constitutive BCR-Mediated Signalling.

B cell antigen receptor (BCR) signalling competence is critical for the pathogenesis of chronic lymphocytic leukaemia (CLL). Defining key proteins that facilitate these networks aid in the identification of targets for therapeutic exploitation. We previously demonstrated that reduced PKCα function in mouse hematopoietic stem/progenitor cells (HPSCs) resulted in PKCßII upregulation and generation of a poor-prognostic CLL-like disease. Here, prkcb knockdown in HSPCs leads to reduced survival of PKCα-KR-expressing CLL-like cells, concurrent with reduced expression of the leukemic markers CD5 and CD23. SP1 promotes elevated expression of prkcb in PKCα-KR expressing cells enabling leukemogenesis. Global gene analysis revealed an upregulation of genes associated with B cell activation in PKCα-KR expressing cells, coincident with upregulation of PKCßII: supported by activation of key signalling hubs proximal to the BCR and elevated proliferation. Ibrutinib (BTK inhibitor) or enzastaurin (PKCßII inhibitor) treatment of PKCα-KR expressing cells and primary CLL cells showed similar patterns of Akt/mTOR pathway inhibition, supporting the role for PKCßII in maintaining proliferative signals in our CLL mouse model. Ibrutinib or enzastaurin treatment also reduced PKCα-KR-CLL cell migration towards CXCL12. Overall, we demonstrate that PKCß expression facilitates leukemogenesis and identify that BCR-mediated signalling is a key driver of CLL development in the PKCα-KR model.

Dasatinib inhibits B cell receptor signalling in chronic lymphocytic leukaemia but novel combination approaches are required to overcome additional pro-survival microenvironmental signals.

As antigenic stimulation of the B cell antigen receptor (BCR) is key to chronic lymphocytic leukaemia (CLL) pathogenesis, targeting dysregulated kinases involved in BCR signalling is an attractive therapeutic approach. We studied the effects of the Src/c-Abl tyrosine kinase inhibitor dasatinib on BCR signal transduction in CLL cells. Treatment of CLL cells with 100 nmol/l dasatinib induced apoptosis by an average reduction in viability of 33·7% at 48 h, with dasatinib sensitivity correlating with inhibition of Syk(Y348) phosphorylation. Dasatinib inhibited calcium flux, phosphatidylinositol-3-kinase and mitogen-activated protein kinase activation following BCR crosslinking, and blocked the Mcl-1-dependent increase in CLL cell survival on prolonged BCR stimulation. However, the pro-apoptotic effect of dasatinib was abrogated by stromal cell contact alone or in the presence of CD154 and interleukin (IL)-4 (CD154L/IL-4 system). Whilst dasatinib retained the ability to sensitize CLL cells in stromal co-culture to both fludarabine and chlorambucil, the addition of CD154 and IL-4 rendered cells resistant to these drug combinations. We demonstrate that the HSP90 inhibitor 17-DMAG exhibited synergy with dasatinib in vitro, and moreover, induced apoptosis of CLL cells in the CD154L/IL-4 system. Our data provide evidence that dasatinib would be most clinically effective in combination with agents able to target antigen-independent microenvironmental signals.

A KDM4A-PAF1-mediated epigenomic network is essential for acute myeloid leukemia cell self-renewal and survival.

Epigenomic dysregulation is a common pathological feature in human hematological malignancies. H3K9me3 emerges as an important epigenomic marker in acute myeloid leukemia (AML). Its associated methyltransferases, such as SETDB1, suppress AML leukemogenesis, whilst H3K9me3 demethylases KDM4C is required for mixed-lineage leukemia rearranged AML. However, the specific role and molecular mechanism of action of another member of the KDM4 family, KDM4A has not previously been clearly defined. In this study, we delineated and functionally validated the epigenomic network regulated by KDM4A. We show that selective loss of KDM4A is sufficient to induce apoptosis in a broad spectrum of human AML cells. This detrimental phenotype results from a global accumulation of H3K9me3 and H3K27me3 at KDM4A targeted genomic loci thereby causing downregulation of a KDM4A-PAF1 controlled transcriptional program essential for leukemogenesis, distinct from that of KDM4C. From this regulatory network, we further extracted a KDM4A-9 gene signature enriched with leukemia stem cell activity; the KDM4A-9 score alone or in combination with the known LSC17 score, effectively stratifies high-risk AML patients. Together, these results establish the essential and unique role of KDM4A for AML self-renewal and survival, supporting further investigation of KDM4A and its targets as a potential therapeutic vulnerability in AML.

ULK1 inhibition promotes oxidative stress-induced differentiation and sensitizes leukemic stem cells to targeted therapy.

Inhibition of autophagy has been proposed as a potential therapy for individuals with cancer. However, current lysosomotropic autophagy inhibitors have demonstrated limited efficacy in clinical trials. Therefore, validation of novel specific autophagy inhibitors using robust preclinical models is critical. In chronic myeloid leukemia (CML), minimal residual disease is maintained by persistent leukemic stem cells (LSCs), which drive tyrosine kinase inhibitor (TKI) resistance and patient relapse. Here, we show that deletion of autophagy-inducing kinase ULK1 (unc-51­like autophagy activating kinase 1) reduces growth of cell line and patient-derived xenografted CML cells in mouse models. Using primitive cells, isolated from individuals with CML, we demonstrate that pharmacological inhibition of ULK1 selectively targets CML LSCs ex vivo and in vivo, when combined with TKI treatment. The enhanced TKI sensitivity after ULK1-mediated autophagy inhibition is driven by increased mitochondrial respiration and loss of quiescence and points to oxidative stress­induced differentiation of CML LSCs, proposing an alternative strategy for treating patients with CML.

A novel transgenic mouse strain expressing PKCßII demonstrates expansion of B1 and marginal zone B cell populations.

Protein kinase Cß (PKCß) expressed in mammalian cells as two splice variants, PKCßI and PKCßII, functions in the B cell receptor (BCR) signaling pathway and contributes to B cell development. We investigated the relative role of PKCßII in B cells by generating transgenic mice where expression of the transgene is directed to these cells using the Eµ promoter (Eµ-PKCßIItg). Our findings demonstrate that homozygous Eµ-PKCßIItg mice displayed a shift from IgD+IgMdim toward IgDdimIgM+ B cell populations in spleen, peritoneum and peripheral blood. Closer examination of these tissues revealed respective expansion of marginal zone (MZ)-like B cells (IgD+IgM+CD43negCD21+CD24+), increased populations of B-1 cells (B220+IgDdimIgM+CD43+CD24+CD5+), and higher numbers of immature B cells (IgDdimIgMdimCD21neg) at the expense of mature B cells (IgD+IgM+CD21+). Therefore, the overexpression of PKCßII, which is a phenotypic feature of chronic lymphocytic leukaemia cells, can skew B cell development in mice, most likely as a result of a regulatory influence on BCR signaling.