Overcoming BCR::ABL1 dependent and independent survival mechanisms in chronic myeloid leukaemia using a multi-kinase targeting approach.

BACKGROUND: Despite improved patient outcome using tyrosine kinase inhibitors (TKIs), chronic myeloid leukaemia (CML) patients require life-long treatment due to leukaemic stem cell (LSC) persistence. LSCs reside in the bone marrow (BM) niche, which they modify to their advantage. The BM provides oncogene-independent signals to aid LSC cell survival and quiescence. The bone-morphogenetic pathway (BMP) is one pathway identified to be highly deregulated in CML, with high levels of BMP ligands detected in the BM, accompanied by CML stem and progenitor cells overexpressing BMP type 1 receptors- activin-like kinases (ALKs), especially in TKI resistant patients. Saracatinib (SC), a SRC/ABL1 dual inhibitor, inhibits the growth of CML cells resistant to the TKI imatinib (IM). Recent studies indicate that SC is also a potent ALK inhibitor and BMP antagonist. Here we investigate the efficacy of SC in overcoming CML BCR::ABL1 dependent and independent signals mediated by the BM niche both in 2D and 3D culture. METHODS: CML cells (K562 cell line and CML CD34+ primary cells) were treated with single or combination treatments of: IM, SC and the BMP receptors inhibitor dorsomorphin (DOR), with or without BMP4 stimulation in 2D (suspension) and 3D co-culture on HS5 stroma cell line and mesenchymal stem cells in AggreWell and microfluidic devices. Flow cytometry was performed to investigate apoptosis, cell cycle progression and proliferation, alongside colony assays following treatment. Proteins changes were validated by immunoblotting and transcriptional changes by Fluidigm multiplex qPCR. RESULTS: By targeting the BMP pathway, using specific inhibitors against ALKs in combination with SRC and ABL TKIs, we show an increase in apoptosis, altered cell cycle regulation, fewer cell divisions, and reduced numbers of CD34+ cells. Impairment of long-term proliferation and differentiation potential after combinatorial treatment also occurred. CONCLUSION: BMP signalling pathway is important for CML cell survival. Targeting SRC, ABL and ALK kinases is more effective than ABL inhibition alone, the combination efficacy importantly being demonstrated in both 2D and 3D cell cultures highlighting the need for combinatorial therapies in contrast to standard of care single agents. Our study provides justification to target multiple kinases in CML to combat LSC persistence.

Blood is made in the spongy inner most section of the bone, called the bone marrow. The bone marrow is where normal blood stem cells live that are responsible for producing the different blood cell types; white blood cells (fight infections), red blood cells (carrying oxygen around the body), platelets (blood clotting) and other cells which support this process. Chronic myeloid leukaemia (CML) is a type of blood cancer that starts in the bone marrow. CML occurs when a normal blood stem cell becomes damaged, forming a leukaemia stem cell (LSC), leading to blood cancer. LSCs multiply and generate many faulty cancerous white blood cells that do not work properly. Patients are treated with a drug called imatinib, which reduces the number of cancerous cells circulating in the body. In many cases, this treatment is not enough to cure the disease because the bone marrow protects the LSCs from the drug meaning patients must remain on long term treatment. This work has discovered one of the ways in which the bone marrow protects LSCs from treatments and has used this knowledge to test new drugs that stop this protection. Our findings show that by combining two drugs, one that overcomes this protection and one that directly targets the cancerous cells, we can destroy more of the LSCs. These findings are a step closer towards a cure for CML and could improve treatment for patients in the future. Video Abstract.

Using topic modeling to detect cellular crosstalk in scRNA-seq.

Cell-cell interactions are vital for numerous biological processes including development, differentiation, and response to inflammation. Currently, most methods for studying interactions on scRNA-seq level are based on curated databases of ligands and receptors. While those methods are useful, they are limited to our current biological knowledge. Recent advances in single cell protocols have allowed for physically interacting cells to be captured, and as such we have the potential to study interactions in a complemantary way without relying on prior knowledge. We introduce a new method based on Latent Dirichlet Allocation (LDA) for detecting genes that change as a result of interaction. We apply our method to synthetic datasets to demonstrate its ability to detect genes that change in an interacting population compared to a reference population. Next, we apply our approach to two datasets of physically interacting cells to identify the genes that change as a result of interaction, examples include adhesion and co-stimulatory molecules which confirm physical interaction between cells. For each dataset we produce a ranking of genes that are changing in subpopulations of the interacting cells. In addition to the genes discussed in the original publications, we highlight further candidates for interaction in the top 100 and 300 ranked genes. Lastly, we apply our method to a dataset generated by a standard droplet-based protocol not designed to capture interacting cells, and discuss its suitability for analysing interactions. We present a method that streamlines detection of interactions and does not require prior clustering and generation of synthetic reference profiles to detect changes in expression.

Inflammatory infiltration is associated with AR expression and poor prognosis in hormone naïve prostate cancer.

BACKGROUND: Tumor microenvironment inflammatory infiltration is proposed as a protumorigenic mechanism for prostate cancer with proinflammatory cytokines stimulating androgen receptor (AR) activity. However, association with patient prognosis remains unclear. This study derives an inflammatory gene signature associated with AR expression and investigates CD3+ and CD8+ T-lymphocyte infiltration association with AR and prognosis. METHODS: Gene profiling of inflammatory related genes was performed on 71 prostate biopsies. Immunohistochemistry on 243 hormone-naïve prostate cancers was performed for CD3, CD8, AR, and phosphorylated AR tumor expression. RESULTS: Multiple proinflammatory genes were differentially expressed in association with high AR expression compared with low AR expression including PI3KCA and MAKP8 (adjusted P < .05). High CD3+ and high CD8+ infiltration associated with reduced cancer-specific survival (P = .018 and P = .020, respectively). High CD3+ infiltration correlated with high tumor cytoplasmic AR expression and if assessed together, they associated with reduced cancer-specific and 5-year survival from 90% to 56% (P = .000179). High CD8+ cytotoxic infiltration associated with high androgen-independent tumor nuclear AR serine 213 phosphorylation (correlation coefficient = 0.227; P = .003) and when assessed together associated with poor clinico-pathological features including perineural invasion (P = .001). Multiple genes involved in proinflammatory signaling pathways are upregulated in high AR expressing prostate samples. CONCLUSION: T-lymphocyte infiltration in hormone-naïve disease associates with androgen-independent driven disease and provides possible therapeutic targets to reduce transformation from hormone-naïve to castrate-resistant disease.

Bone marrow niche crosses paths with BMPs: a road to protection and persistence in CML.

Chronic myeloid leukaemia (CML) is a paradigm of precision medicine, being one of the first cancers to be treated with targeted therapy. This has revolutionised CML therapy and patient outcome, with high survival rates. However, this now means an ever-increasing number of patients are living with the disease on life-long tyrosine kinase inhibitor (TKI) therapy, with most patients anticipated to have near normal life expectancy. Unfortunately, in a significant number of patients, TKIs are not curative. This low-level disease persistence suggests that despite a molecularly targeted therapeutic approach, there are BCR-ABL1-independent mechanisms exploited to sustain the survival of a small cell population of leukaemic stem cells (LSCs). In CML, LSCs display many features akin to haemopoietic stem cells, namely quiescence, self-renewal and the ability to produce mature progeny, this all occurs through intrinsic and extrinsic signals within the specialised microenvironment of the bone marrow (BM) niche. One important avenue of investigation in CML is how the disease highjacks the BM, thereby remodelling this microenvironment to create a niche, which enables LSC persistence and resistance to TKI treatment. In this review, we explore how changes in growth factor levels, in particular, the bone morphogenetic proteins (BMPs) and pro-inflammatory cytokines, impact on cell behaviour, extracellular matrix deposition and bone remodelling in CML. We also discuss the challenges in targeting LSCs and the potential of dual targeting using combination therapies against BMP receptors and BCR-ABL1.

Role of the bone morphogenic protein pathway in developmental haemopoiesis and leukaemogenesis.

Myeloid leukaemias share the common characteristics of being stem cell-derived clonal diseases, characterised by excessive proliferation of one or more myeloid lineage. Chronic myeloid leukaemia (CML) arises from a genetic alteration in a normal haemopoietic stem cell (HSC) giving rise to a leukaemic stem cell (LSC) within the bone marrow (BM) 'niche'. CML is characterised by the presence of the oncogenic tyrosine kinase fusion protein breakpoint cluster region-abelson murine leukaemia viral oncogene homolog 1 (BCR-ABL), which is responsible for driving the disease through activation of downstream signal transduction pathways. Recent evidence from our group and others indicates that important regulatory networks involved in establishing primitive and definitive haemopoiesis during development are reactivated in myeloid leukaemia, giving rise to an LSC population with altered self-renewal and differentiation properties. In this review, we explore the role the bone morphogenic protein (BMP) signalling plays in stem cell pluripotency, developmental haemopoiesis, HSC maintenance and the implication of altered BMP signalling on LSC persistence in the BM niche. Overall, we emphasise how the BMP and Wnt pathways converge to alter the Cdx-Hox axis and the implications of this in the pathogenesis of myeloid malignancies.

JAK2/STAT5 inhibition by nilotinib with ruxolitinib contributes to the elimination of CML CD34+ cells in vitro and in vivo.

Chronic myeloid leukemia (CML) stem cell survival is not dependent on BCR-ABL protein kinase and treatment with ABL tyrosine kinase inhibitors cures only a minority of CML patients, thus highlighting the need for novel therapeutic targets. The Janus kinase (JAK)2/signal transducer and activator of transcription (STAT)5 pathway has recently been explored for providing putative survival signals to CML stem/progenitor cells (SPCs) with contradictory results. We investigated the role of this pathway using the JAK2 inhibitor, ruxolitinib (RUX). We demonstrated that the combination of RUX, at clinically achievable concentrations, with the specific and potent tyrosine kinase inhibitor nilotinib, reduced the activity of the JAK2/STAT5 pathway in vitro relative to either single agent alone. These effects correlated with increased apoptosis of CML SPCs in vitro and a reduction in primitive quiescent CML stem cells, including NOD.Cg-Prkdc(scid) IL2rg(tm1Wjl) /SzJ mice repopulating cells, induced by combination treatment. A degree of toxicity toward normal SPCs was observed with the combination treatment, although this related to mature B-cell engraftment in NOD.Cg-Prkdc(scid) IL2rg(tm1Wjl) /SzJ mice with minimal effects on primitive CD34(+) cells. These results support the JAK2/STAT5 pathway as a relevant therapeutic target in CML SPCs and endorse the current use of nilotinib in combination with RUX in clinical trials to eradicate persistent disease in CML patients.

Acute loss of Cited2 impairs Nanog expression and decreases self-renewal of mouse embryonic stem cells.

Identifying novel players of the pluripotency gene regulatory network centered on Oct4, Sox2, and Nanog as well as delineating the interactions within the complex network is key to understanding self-renewal and early cell fate commitment of embryonic stem cells (ESC). While overexpression of the transcriptional regulator Cited2 sustains ESC pluripotency, its role in ESC functions remains unclear. Here, we show that Cited2 is important for proliferation, survival, and self-renewal of mouse ESC. We position Cited2 within the pluripotency gene regulatory network by defining Nanog, Tbx3, and Klf4 as its direct targets. We also demonstrate that the defects caused by Cited2 depletion are, at least in part, rescued by Nanog constitutive expression. Finally, we demonstrate that Cited2 is required for and enhances reprogramming of mouse embryonic fibroblasts to induced pluripotent stem cells.

The role of the bone morphogenetic proteins in leukaemic stem cell persistence.

CML (chronic myeloid leukaemia) is characterized by the presence of the oncogenic tyrosine kinase fusion protein BCR (breakpoint cluster region)-Abl, responsible for driving the disease. Current TKI (tyrosine kinase inhibitor) therapies effectively inhibit BCR-Abl to control CML in the majority of patients, but do not eliminate the LSC (leukaemic stem cell) population, which becomes quiescent following treatment. Patients require long-term treatment to sustain remission; alternative strategies are therefore required, either alone or in combination with TKIs to eliminate the LSCs and provide a cure. The embryonic morphogenetic pathways play a key role in haemopoiesis with recent evidence suggesting LSCs are more dependent on these signals following chemotherapy than normal HSCs (haemopoietic stem cells). Recent evidence in the literature and from our group has revealed that the BMP (bone morphogenetic protein) pathway is differentially expressed in CML patients compared with normal donors. In the present review, we explore the role that BMP signalling plays in oesteoblast differentiation, HSC maintenance and the implication of altered BMP signalling on LSC persistence in the BM (bone marrow) niche. Overall, we highlight the BMP pathway as a potential target for developing LSC-directed therapies in CML in the future.

Complexities of modeling the bone marrow microenvironment to facilitate hematopoietic research.

Hematopoiesis occurs in the bone marrow (BM), within a specialized microenvironment referred to as the stem cell niche, where the hematopoietic stem cells (HSCs) reside and are regulated for quiescence, self-renewal and differentiation through intrinsic and extrinsic mechanisms. The BM contains at least two distinctive HSC-supportive niches: an endosteal osteoblastic niche that supports quiescence and self-renewal and a more vascular/perisinusoidal niche that promotes proliferation and differentiation. Both associate with supporting mesenchymal stromal cells. Within the more hypoxic osteoblastic niche, HSCs specifically interact with the osteoblasts that line the endosteal surface, which secrete several important HSC quiescence and maintenance regulatory factors. In vivo imaging indicates that the HSCs and progenitors located further away, in the vicinity of sinusoidal endothelial cells, are more proliferative. Here, HSCs interact with endothelial cells via specific cell adhesion molecules. Endothelial cells also secrete several factors important for HSC homeostasis and proliferation. In addition, HSCs and mesenchymal stromal cells are embedded within the extracellular matrix (ECM), an important network of proteins such as collagen, elastin, laminin, proteoglycans, vitronectin, and fibronectin. The ECM provides mechanical characteristics such as stiffness and elasticity important for cell behavior regulation. ECM proteins are also able to bind, sequester, display, and distribute growth factors across the BM, thus directly affecting stem cell fate and regulation of hematopoiesis. These important physical and chemical features of the BM require careful consideration when creating three-dimensional models of the BM.

Bioengineered niches that recreate physiological extracellular matrix organisation to support long-term haematopoietic stem cells.

Long-term reconstituting haematopoietic stem cells (LT-HSCs) are used to treat blood disorders via stem cell transplantation. The very low abundance of LT-HSCs and their rapid differentiation during in vitro culture hinders their clinical utility. Previous developments using stromal feeder layers, defined media cocktails, and bioengineering have enabled HSC expansion in culture, but of mostly short-term HSCs and progenitor populations at the expense of naive LT-HSCs. Here, we report the creation of a bioengineered LT-HSC maintenance niche that recreates physiological extracellular matrix organisation, using soft collagen type-I hydrogels to drive nestin expression in perivascular stromal cells (PerSCs). We demonstrate that nestin, which is expressed by HSC-supportive bone marrow stromal cells, is cytoprotective and, via regulation of metabolism, is important for HIF-1α expression in PerSCs. When CD34+ve HSCs were added to the bioengineered niches comprising nestin/HIF-1α expressing PerSCs, LT-HSC numbers were maintained with normal clonal and in vivo reconstitution potential, without media supplementation. We provide proof-of-concept that our bioengineered niches can support the survival of CRISPR edited HSCs. Successful editing of LT-HSCs ex vivo can have potential impact on the treatment of blood disorders.

Leukaemia exposure alters the transcriptional profile and function of BCR::ABL1 negative macrophages in the bone marrow niche.

Macrophages are fundamental cells of the innate immune system that support normal haematopoiesis and play roles in both anti-cancer immunity and tumour progression. Here we use a chimeric mouse model of chronic myeloid leukaemia (CML) and human bone marrow (BM) derived macrophages to study the impact of the dysregulated BM microenvironment on bystander macrophages. Utilising single-cell RNA sequencing (scRNA-seq) of Philadelphia chromosome (Ph) negative macrophages we reveal unique subpopulations of immature macrophages residing in the CML BM microenvironment. CML exposed macrophages separate from their normal counterparts by reduced expression of the surface marker CD36, which significantly reduces clearance of apoptotic cells. We uncover aberrant production of CML-secreted factors, including the immune modulatory protein lactotransferrin (LTF), that suppresses efferocytosis, phagocytosis, and CD36 surface expression in BM macrophages, indicating that the elevated secretion of LTF is, at least partially responsible for the supressed clearance function of Ph- macrophages.

Targeting self-renewal pathways in myeloid malignancies.

A fundamental property of hematopoietic stem cells (HSCs) is the ability to self-renew. This is a complex process involving multiple signal transduction cascades which control the fine balance between self-renewal and differentiation through transcriptional networks. Key activators/regulators of self-renewal include chemokines, cytokines and morphogens which are expressed in the bone marrow niche, either in a paracrine or autocrine fashion, and modulate stem cell behaviour. Increasing evidence suggests that the downstream signaling pathways induced by these ligands converge at multiple levels providing a degree of redundancy in steady state hematopoiesis. Here we will focus on how these pathways cross-talk to regulate HSC self-renewal highlighting potential therapeutic windows which could be targeted to prevent leukemic stem cell self-renewal in myeloid malignancies.

Micro-environment alterations through time leading to myeloid malignancies.

The micro-environment plays a critical role in haematopoietic stem cell (HSC) development, self-renewal, differentiation and maintenance by providing a supportive cellular framework and essential molecular cues to sustain homeostasis. In ageing and development of age-related clonal haematopoiesis, the combined contribution of intrinsic alterations in haematopoietic stem cells and their surrounding micro-environment can promote myeloid skewing and release of pro-inflammatory cytokines. A pro-inflammatory micro-environment is a common feature in the initiation and sustenance of several myeloid malignancies. Furthermore, remodelling of the micro-environment is recognized to potentiate the survival of malignant over normal cells. This review explores micro-environmental interactions in the haematopoietic system of adults, especially how the bone marrow micro-environment is impacted by ageing, the onset of age-related clonal haematopoiesis and the development of myeloid malignancies. In addition, we also discuss the possible role age-related clonal haematopoiesis and chronic inflammatory conditions play in altering the bone marrow micro-environment dynamics. Finally, we explore the importance of in vitro models that accurately mimic different aspects of the bone marrow micro-environment in order to study normal and malignant haematopoiesis.

BH3 mimetics in combination with nilotinib or ponatinib represent a promising therapeutic strategy in blast phase chronic myeloid leukemia.

Dysregulation of the BCL-2 family is implicated in protecting chronic myeloid leukemia (CML) cells from intracellular damage and BCR::ABL1-inhibition with tyrosine kinase inhibitors (TKIs) and may be a viable therapeutic target in blast phase (BP-)CML, for which treatment options are limited. BH3 mimetics, a class of small molecule inhibitors with high-specificity against the prosurvival members of the BCL-2 family, have displayed clinical promise in the treatment of chronic lymphocytic and acute myeloid leukemia as single agents and in combination with standard-of-care therapies. Here we present the first comparison of inhibition of BCL-2 prosurvival proteins BCL-2, BCL-xL and MCL-1 in combination with a second or third generation TKI, crucially with comparisons drawn between myeloid and lymphoid BP-CML samples. Co-treatment of four BP-CML cell lines with the TKIs nilotinib or ponatinib and either BCL-2 (venetoclax), MCL-1 (S63845) or BCL-xL (A-1331852) inhibitors resulted in a synergistic reduction in cell viability and increase in phosphatidylserine (PS) presentation. Nilotinib with BH3 mimetic combinations in myeloid BP-CML patient samples triggered increased induction of apoptosis over nilotinib alone, and a reduction in colony-forming capacity and CD34+ fraction, while this was not the case for lymphoid BP-CML samples tested. While some heterogeneity in apoptotic response was observed between cell lines and BP-CML patient samples, the combination of BCL-xL and BCR::ABL1 inhibition was consistently effective in inducing substantial apoptosis. Further, while BH3 mimetics showed little efficacy as single agents, dual-inhibition of BCL-2 prosurvival proteins dramatically induced apoptosis in all cell lines tested and in myeloid BP-CML patient samples compared to healthy donor samples. Gene expression and protein level analysis suggests a protective upregulation of alternative BCL-2 prosurvival proteins in response to BH3 mimetic single-treatment in BP-CML. Our results suggest that BH3 mimetics represent an interesting avenue for further exploration in myeloid BP-CML, for which alternative treatment options are desperately sought.

The application of BH3 mimetics in myeloid leukemias.

Execution of the intrinsic apoptotic pathway is controlled by the BCL-2 proteins at the level of the mitochondrial outer membrane (MOM). This family of proteins consists of prosurvival (e.g., BCL-2, MCL-1) and proapoptotic (e.g., BIM, BAD, HRK) members, the functional balance of which dictates the activation of BAX and BAK. Once activated, BAX/BAK form pores in the MOM, resulting in cytochrome c release from the mitochondrial intermembrane space, leading to apoptosome formation, caspase activation, and cleavage of intracellular targets. This pathway is induced by cellular stress including DNA damage, cytokine and growth factor withdrawal, and chemotherapy/drug treatment. A well-documented defense of leukemia cells is to shift the balance of the BCL-2 family in favor of the prosurvival proteins to protect against such intra- and extracellular stimuli. Small molecule inhibitors targeting the prosurvival proteins, named 'BH3 mimetics', have come to the fore in recent years to treat hematological malignancies, both as single agents and in combination with standard-of-care therapies. The most significant example of these is the BCL-2-specific inhibitor venetoclax, given in combination with standard-of-care therapies with great success in AML in clinical trials. As the number and variety of available BH3 mimetics increases, and investigations into applying these novel inhibitors to treat myeloid leukemias continue apace the need to evaluate where we currently stand in this rapidly expanding field is clear.

Macrophages in Acute Myeloid Leukaemia: Significant Players in Therapy Resistance and Patient Outcomes.

Acute Myeloid Leukaemia (AML) is a commonly occurring severe haematological malignancy, with most patients exhibiting sub-optimal clinical outcomes. Therapy resistance significantly contributes towards failure of traditional and targeted treatments, disease relapse and mortality in AML patients. The mechanisms driving therapy resistance in AML are not fully understood, and approaches to overcome therapy resistance are important for curative therapies. To date, most studies have focused on therapy resistant mechanisms inherent to leukaemic cells (e.g., TP53 mutations), overlooking to some extent, acquired mechanisms of resistance through extrinsic processes. In the bone marrow microenvironment (BMME), leukaemic cells interact with the surrounding bone resident cells, driving acquired therapy resistance in AML. Growing evidence suggests that macrophages, highly plastic immune cells present in the BMME, play a role in the pathophysiology of AML. Leukaemia-supporting macrophage subsets (CD163+CD206+) are elevated in preclinical in vivo models of AML and AML patients. However, the relationship between macrophages and therapy resistance in AML warrants further investigation. In this review, we correlate the potential links between macrophages, the development of therapy resistance, and patient outcomes in AML. We specifically focus on macrophage reprogramming by AML cells, macrophage-driven activation of anti-cell death pathways in AML cells, and the association between macrophage phenotypes and clinical outcomes in AML, including their potential prognostic value. Lastly, we discuss therapeutic targeting of macrophages, as a strategy to circumvent therapy resistance in AML, and discuss how emerging genomic and proteomic-based approaches can be utilised to address existing challenges in this research field.

Interrogation of novel CDK2/9 inhibitor fadraciclib (CYC065) as a potential therapeutic approach for AML.

Over the last 50 years, there has been a steady improvement in the treatment outcome of acute myeloid leukemia (AML). However, median survival in the elderly is still poor due to intolerance to intensive chemotherapy and higher numbers of patients with adverse cytogenetics. Fadraciclib (CYC065), a novel cyclin-dependent kinase (CDK) 2/9 inhibitor, has preclinical efficacy in AML. In AML cell lines, myeloid cell leukemia 1 (MCL-1) was downregulated following treatment with fadraciclib, resulting in a rapid induction of apoptosis. In addition, RNA polymerase II (RNAPII)-driven transcription was suppressed, rendering a global gene suppression. Rapid induction of apoptosis was observed in primary AML cells after treatment with fadraciclib for 6-8 h. Twenty-four hours continuous treatment further increased efficacy of fadraciclib. Although preliminary results showed that AML cell lines harboring KMT2A rearrangement (KMT2A-r) are more sensitive to fadraciclib, we found that the drug can induce apoptosis and decrease MCL-1 expression in primary AML cells, regardless of KMT2A status. Importantly, the diversity of genetic mutations observed in primary AML patient samples was associated with variable response to fadraciclib, confirming the need for patient stratification to enable a more effective and personalized treatment approach. Synergistic activity was demonstrated when fadraciclib was combined with the BCL-2 inhibitor venetoclax, or the conventional chemotherapy agents, cytarabine, or azacitidine, with the combination of fadraciclib and azacitidine having the most favorable therapeutic window. In summary, these results highlight the potential of fadraciclib as a novel therapeutic approach for AML.

CD93 is expressed on chronic myeloid leukemia stem cells and identifies a quiescent population which persists after tyrosine kinase inhibitor therapy.

The introduction of BCR-ABL tyrosine kinase inhibitors has revolutionized the treatment of chronic myeloid leukemia (CML). A major clinical aim remains the identification and elimination of low-level disease persistence, termed "minimal residual disease". The phenomenon of disease persistence suggests that despite targeted therapeutic approaches, BCR-ABL-independent mechanisms exist which sustain the survival of leukemic stem cells (LSCs). Although other markers of a primitive CML LSC population have been identified in the preclinical setting, only CD26 appears to offer clinical utility. Here we demonstrate consistent and selective expression of CD93 on a lin-CD34+CD38-CD90+ CML LSC population and show in vitro and in vivo data to suggest increased stem cell characteristics, as well as robust engraftment in patient-derived xenograft models in comparison with a CD93- CML stem/progenitor cell population, which fails to engraft. Through bulk and single-cell analyses of selected stem cell and cell survival-specific genes, we confirmed the quiescent character and demonstrate their persistence in a population of CML patient samples who demonstrate molecular relapse on TKI withdrawal. Taken together, our results identify that CD93 is consistently and selectively expressed on a lin-CD34+CD38-CD90+ CML LSC population with stem cell characteristics and may be an important indicator in determining poor TKI responders.

Correction: CD93 is expressed on chronic myeloid leukemia stem cells and identifies a quiescent population which persists after tyrosine kinase inhibitor therapy.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Chronic myeloid leukaemia cells require the bone morphogenic protein pathway for cell cycle progression and self-renewal.

Leukaemic stem cell (LSC) persistence remains a major obstacle to curing chronic myeloid leukaemia (CML). The bone morphogenic protein (BMP) pathway is deregulated in CML, with altered expression and response to the BMP ligands shown to impact on LSC expansion and behaviour. In this study, we determined whether alterations in the BMP pathway gene signature had any predictive value for therapeutic response by profiling 60 CML samples at diagnosis from the UK SPIRIT2 trial and correlating the data to treatment response using the 18-month follow-up data. There was significant deregulation of several genes involved in the BMP pathway with ACV1C, INHBA, SMAD7, SNAIL1 and SMURF2 showing differential expression in relation to response. Therapeutic targeting of CML cells using BMP receptor inhibitors, in combination with tyrosine kinase inhibitor (TKI), indicate a synergistic mode of action. Furthermore, dual treatment resulted in altered cell cycle gene transcription and irreversible cell cycle arrest, along with increased apoptosis compared to single agents. Targeting CML CD34+ cells with BMP receptor inhibitors resulted in fewer cell divisions, reduced numbers of CD34+ cells and colony formation when compared to normal donor CD34+ cells, both in the presence and absence of BMP4. In an induced pluripotent stem cell (iPSC) model generated from CD34+ hematopoietic cells, we demonstrate altered cell cycle profiles and dynamics of ALK expression in CML-iPSCs in the presence and absence of BMP4 stimulation, when compared to normal iPSC. Moreover, dual targeting with TKI and BMP inhibitor prevented the self-renewal of CML-iPSC and increased meso-endodermal differentiation. These findings indicate that transformed stem cells may be more reliant on BMP signalling than normal stem cells. These changes offer a therapeutic window in CML, with intervention using BMP inhibitors in combination with TKI having the potential to target LSC self-renewal and improve long-term outcome for patients.