Arginine dependency is a therapeutically exploitable vulnerability in chronic myeloid leukaemic stem cells.

To fuel accelerated proliferation, leukaemic cells undergo metabolic deregulation, which can result in specific nutrient dependencies. Here, we perform an amino acid drop-out screen and apply pre-clinical models of chronic phase chronic myeloid leukaemia (CML) to identify arginine as a nutrient essential for primary human CML cells. Analysis of the Microarray Innovations in Leukaemia (MILE) dataset uncovers reduced ASS1 levels in CML compared to most other leukaemia types. Stable isotope tracing reveals repressed activity of all urea cycle enzymes in patient-derived CML CD34+ cells, rendering them arginine auxotrophic. Thus, arginine deprivation completely blocks proliferation of CML CD34+ cells and induces significantly higher levels of apoptosis when compared to arginine-deprived cell lines. Similarly, primary CML cells, but not normal CD34+ samples, are particularly sensitive to treatment with the arginine-depleting enzyme, BCT-100, which induces apoptosis and reduces clonogenicity. Moreover, BCT-100 is highly efficacious in a patient-derived xenograft model, causing > 90% reduction in the number of human leukaemic stem cells (LSCs). These findings indicate arginine depletion to be a promising and novel strategy to eradicate therapy resistant LSCs.

A randomised evaluation of low-dose cytosine arabinoside plus lenalidomide versus single-agent low-dose cytosine arabinoside in older patients with acute myeloid leukaemia: Results from the LI-1 trial.

Improving outcomes for older patients with acute myeloid leukaemia remains an unmet need. As part of the LI-1 trial, we evaluated lenalidomide (LEN) in combination with low-dose cytosine arabinoside (LDAC) in patients aged >60 years unfit for intensive therapy and compared this to LDAC alone. Two hundred and two patients, randomised 1:1, were evaluable. Overall response rate (CR + CRi) was higher for LDAC + LEN versus LDAC (26% and 13.7% respectively p = 0.031). However, there was no difference in overall survival between the arms (14% and 11.5% at 2 years for LDAC + LEN and LDAC respectively). The addition of LEN was associated with increased toxicity and supportive care requirements.

A randomised evaluation of low-dose Ara-C plus pegylated recombinant arginase BCT-100 versus low dose Ara-C in older unfit patients with acute myeloid leukaemia: Results from the LI-1 trial.

The survival of acute myeloid leukaemia (AML) patients aged over 60 has been suboptimal historically, whether they are treated using hypomethylating agents, low-dose cytarabine (LDAC) or venetoclax-based regimens. Progress is being made, however, for subgroups with favourable molecular or cytogenetic findings. Arginine metabolism plays a key role in AML pathophysiology. We report the only randomised study of LDAC with recombinant arginase BCT-100 versus LDAC alone in older AML patients unsuitable for intensive therapy. Eighty-three patients were randomised to the study. An overall response rate was seen in 19.5% (all complete remission [CR]) and 15% (7.5% each in CR and CR without evidence of adequate count recovery [CRi]) of patients in the LDAC+BCT-100 and LDAC arms respectively (odds ratio 0.73, confidence interval 0.23-2.33; p = 0.592). No significant difference in overall or median survival between treatment arms was seen. The addition of BCT-100 to LDAC was well tolerated.

Treatment of blast phase chronic myeloid leukaemia: A rare and challenging entity.

Despite the success of BCR-ABL-specific tyrosine kinase inhibitors (TKIs) such as imatinib in chronic phase (CP) chronic myeloid leukaemia (CML), patients with blast phase (BP)-CML continue to have a dismal outcome with median survival of less than one year from diagnosis. Thus BP-CML remains a critical unmet clinical need in the management of CML. Our understanding of the biology of BP-CML continues to grow; genomic instability leads to acquisition of mutations which drive leukaemic progenitor cells to develop self-renewal properties, resulting in differentiation block and a poor-prognosis acute leukaemia which may be myeloid, lymphoid or bi-phenotypic. Similar advances in therapy are urgently needed to improve patient outcomes; however, this is challenging given the rarity and heterogeneity of BP-CML, leading to difficulty in designing and recruiting to prospective clinical trials. This review will explore the treatment of BP-CML, evaluating the data for TKI therapy alone, combinations with intensive chemotherapy, the role of allogeneic haemopoietic stem cell transplantation, the use of novel agents and clinical trials, as well as discussing the most appropriate methods for diagnosing BP and assessing response to therapy, and factors predicting outcome.

Dual targeting of p53 and c-MYC selectively eliminates leukaemic stem cells.

Chronic myeloid leukaemia (CML) arises after transformation of a haemopoietic stem cell (HSC) by the protein-tyrosine kinase BCR-ABL. Direct inhibition of BCR-ABL kinase has revolutionized disease management, but fails to eradicate leukaemic stem cells (LSCs), which maintain CML. LSCs are independent of BCR-ABL for survival, providing a rationale for identifying and targeting kinase-independent pathways. Here we show--using proteomics, transcriptomics and network analyses--that in human LSCs, aberrantly expressed proteins, in both imatinib-responder and non-responder patients, are modulated in concert with p53 (also known as TP53) and c-MYC regulation. Perturbation of both p53 and c-MYC, and not BCR-ABL itself, leads to synergistic cell kill, differentiation, and near elimination of transplantable human LSCs in mice, while sparing normal HSCs. This unbiased systems approach targeting connected nodes exemplifies a novel precision medicine strategy providing evidence that LSCs can be eradicated.

A randomised evaluation of low-dose cytosine arabinoside (ara-C) plus tosedostat versus low-dose ara-C in older patients with acute myeloid leukaemia: results of the LI-1 trial.

Older patients with acute myeloid leukaemia (AML) account for nearly half of those with the disease. Because they are perceived to be unfit for, unwilling to receive, or unlikely to benefit from conventional chemotherapy they represent an important unmet need. Tosedostat is a selective oral aminopeptidase inhibitor, which in phase I/II trials showed acceptable toxicity and encouraging efficacy. We report the only randomised study of low-dose cytosine arabinoside (LDAC) combined with tosedostat (LDAC-T) versus LDAC in untreated older patients not suitable for intensive treatment. A total of 243 patients were randomised 1:1 as part of the 'Pick-a-Winner' LI-1 trial. There was a statistically non-significant increase in the complete remission (CR) rate with the addition of tosedostat, LDAC-T 19% versus LDAC 12% [odds ratio (OR) 0·61, 95% confidence interval (CI) 0·30-1·23; P = 0·17]. For overall response (CR+CR with incomplete recovery of counts), there was little evidence of a benefit to the addition of tosedostat (25% vs. 18%; OR 0·68, 95% CI 0·37-1·27; P = 0·22). However, overall survival (OS) showed no difference (2-year OS 16% vs. 12%, hazard ratio 0·97, 95% CI 0·73-1·28; P = 0·8). Exploratory analyses failed to identify any subgroup benefitting from tosedostat. Despite promising pre-clinical, early non-randomised clinical data with acceptable toxicity and an improvement in response, we did not find evidence that the addition of tosedostat to LDAC produced a survival benefit in this group of patients with AML. International Standard Randomised Controlled Trial Number: ISRCTN40571019.

Clonal haematopoiesis of indeterminate potential: intersections between inflammation, vascular disease and heart failure.

Ageing is a major risk factor for the development of cardiovascular disease (CVD) and cancer. Whilst the cumulative effect of exposure to conventional cardiovascular risk factors is important, recent evidence highlights clonal haematopoiesis of indeterminant potential (CHIP) as a further key risk factor. CHIP reflects the accumulation of somatic, potentially pro-leukaemic gene mutations within haematopoietic stem cells over time. The most common mutations associated with CHIP and CVD occur in genes that also play central roles in the regulation of inflammation. While CHIP carriers have a low risk of haematological malignant transformation (<1% per year), their relative risk of mortality is increased by 40% and this reflects an excess of cardiovascular events. Evidence linking CHIP, inflammation and atherosclerotic disease has recently become better defined. However, there is a paucity of information about the role of CHIP in the development and progression of heart failure, particularly heart failure with preserved ejection fraction (HFpEF). While systemic inflammation plays a role in the pathophysiology of both heart failure with reduced and preserved ejection fraction (EF), it may be of greater relevance in the pathophysiology of HFpEF, which is also strongly associated with ageing. This review describes CHIP and its pathogenetic links with ageing, inflammation and CVD, while providing insight into its putative role in HFpEF.

Consensus on BCR-ABL1 reporting in chronic myeloid leukaemia in the UK.

For patients with chronic myeloid leukaemia (CML), treatment guidelines recommend monitoring response to treatment with tyrosine kinase inhibitors (TKIs) by testing the BCR-ABL1 fusion gene transcript level using reverse transcriptase quantitative polymerase chain reaction. Despite recent efforts to standardise protocols for BCR-ABL1 testing, some variability remains among laboratories in the UK regarding the techniques used and the approach to reporting results. This increases the risk of misinterpretation of results by both clinicians and patients. An expert panel met to discuss current issues surrounding BCR-ABL1 testing in the UK and to develop guidance for laboratories, with emphasis on the optimal approach to reporting laboratory results. Topics included the minimum required information to include in the laboratory report, units of measurement, test sensitivity and BCR-ABL1 transcript variants. To aid communication between laboratories and clinics, standard forms were generated that could be used by (i) clinics when submitting samples to laboratories, and (ii) laboratories when reporting results to clinics. Standardising the way in which BCR-ABL1 test results are reported from laboratories to clinics should help to improve communication, interpretation of results and patient care.

What are the challenges in 2016 regarding resistance to tyrosine kinase inhibitors in chronic myeloid leukemia and cancer?

In the past decade, the treatment of chronic myeloid leukemia (CML) has undergone a drastic evolution. The discovery and success of imatinib and second-generation tyrosine kinase inhibitors have substantially increased the outcome for CML patients. The next step in medical and scientific research is to better understand the malignancy so as to eventually find a cure to eliminate all leukemic cells from patients. One of the key issues is about the resistance of the leukemic stem cells to tyrosine kinase inhibitors. Here, we briefly describe our current studies on CML resistance, and leukemic stem cell modeling and characterization.

Implementing the EffTox dose-finding design in the Matchpoint trial.

BACKGROUND: The Matchpoint trial aims to identify the optimal dose of ponatinib to give with conventional chemotherapy consisting of fludarabine, cytarabine and idarubicin to chronic myeloid leukaemia patients in blastic transformation phase. The dose should be both tolerable and efficacious. This paper describes our experience implementing EffTox in the Matchpoint trial. METHODS: EffTox is a Bayesian adaptive dose-finding trial design that jointly scrutinises binary efficacy and toxicity outcomes. We describe a nomenclature for succinctly describing outcomes in phase I/II dose-finding trials. We use dose-transition pathways, where doses are calculated for each feasible set of outcomes in future cohorts. We introduce the phenomenon of dose ambivalence, where EffTox can recommend different doses after observing the same outcomes. We also describe our experiences with outcome ambiguity, where the categorical evaluation of some primary outcomes is temporarily delayed. RESULTS: We arrived at an EffTox parameterisation that is simulated to perform well over a range of scenarios. In scenarios where dose ambivalence manifested, we were guided by the dose-transition pathways. This technique facilitates planning, and also helped us overcome short-term outcome ambiguity. CONCLUSIONS: EffTox is an efficient and powerful design, but not without its challenges. Joint phase I/II clinical trial designs will likely become increasingly important in coming years as we further investigate non-cytotoxic treatments and streamline the drug approval process. We hope this account of the problems we faced and the solutions we used will help others implement this dose-finding clinical trial design. TRIAL REGISTRATION: Matchpoint was added to the European Clinical Trials Database ( https://www.clinicaltrialsregister.eu/ctr-search/trial/2012-005629-65/GB ) on 2013-12-30.

Autocrine TNF-α production supports CML stem and progenitor cell survival and enhances their proliferation.

Chronic myeloid leukemia (CML) stem cells are not dependent on BCR-ABL kinase for their survival, suggesting that kinase-independent mechanisms must contribute to their persistence. We observed that CML stem/progenitor cells (SPCs) produce tumor necrosis factor-α (TNF-α) in a kinase-independent fashion and at higher levels relative to their normal counterparts. We therefore investigated the role of TNF-α and found that it supports survival of CML SPCs by promoting nuclear factor κB/p65 pathway activity and expression of the interleukin 3 and granulocyte/macrophage-colony stimulating factor common ß-chain receptor. Furthermore, we demonstrate that in CML SPCs, inhibition of autocrine TNF-α signaling via a small-molecule TNF-α inhibitor induces apoptosis. Moreover TNF-α inhibition combined with nilotinib induces significantly more apoptosis relative to either treatment alone and a reduction in the absolute number of primitive quiescent CML stem cells. These results highlight a novel survival mechanism of CML SPCs and suggest a new putative therapeutic target for their eradication.

Personalized synthetic lethality induced by targeting RAD52 in leukemias identified by gene mutation and expression profile.

Homologous recombination repair (HRR) protects cells from the lethal effect of spontaneous and therapy-induced DNA double-stand breaks. HRR usually depends on BRCA1/2-RAD51, and RAD52-RAD51 serves as back-up. To target HRR in tumor cells, a phenomenon called "synthetic lethality" was applied, which relies on the addiction of cancer cells to a single DNA repair pathway, whereas normal cells operate 2 or more mechanisms. Using mutagenesis and a peptide aptamer approach, we pinpointed phenylalanine 79 in RAD52 DNA binding domain I (RAD52-phenylalanine 79 [F79]) as a valid target to induce synthetic lethality in BRCA1- and/or BRCA2-deficient leukemias and carcinomas without affecting normal cells and tissues. Targeting RAD52-F79 disrupts the RAD52-DNA interaction, resulting in the accumulation of toxic DNA double-stand breaks in malignant cells, but not in normal counterparts. In addition, abrogation of RAD52-DNA interaction enhanced the antileukemia effect of already-approved drugs. BRCA-deficient status predisposing to RAD52-dependent synthetic lethality could be predicted by genetic abnormalities such as oncogenes BCR-ABL1 and PML-RAR, mutations in BRCA1 and/or BRCA2 genes, and gene expression profiles identifying leukemias displaying low levels of BRCA1 and/or BRCA2. We believe this work may initiate a personalized therapeutic approach in numerous patients with tumors displaying encoded and functional BRCA deficiency.

The antiproliferative activity of kinase inhibitors in chronic myeloid leukemia cells is mediated by FOXO transcription factors.

Chronic myeloid leukemia (CML) is initiated and maintained by the tyrosine kinase BCR-ABL which activates a number of signal transduction pathways, including PI3K/AKT signaling and consequently inactivates FOXO transcription factors. ABL-specific tyrosine kinase inhibitors (TKIs) induce minimal apoptosis in CML progenitor cells, yet exert potent antiproliferative effects, through as yet poorly understood mechanisms. Here, we demonstrate that in CD34+ CML cells, FOXO1 and 3a are inactivated and relocalized to the cytoplasm by BCR-ABL activity. TKIs caused a decrease in phosphorylation of FOXOs, leading to their relocalization from cytoplasm (inactive) to nucleus (active), where they modulated the expression of key FOXO target genes, such as Cyclin D1, ATM, CDKN1C, and BCL6 and induced G1 arrest. Activation of FOXO1 and 3a and a decreased expression of their target gene Cyclin D1 were also observed after 6 days of in vivo treatment with dasatinib in a CML transgenic mouse model. The over-expression of FOXO3a in CML cells combined with TKIs to reduce proliferation, with similar results seen for inhibitors of PI3K/AKT/mTOR signaling. While stable expression of an active FOXO3a mutant induced a similar level of quiescence to TKIs alone, shRNA-mediated knockdown of FOXO3a drove CML cells into cell cycle and potentiated TKI-induced apoptosis. These data demonstrate that TKI-induced G1 arrest in CML cells is mediated through inhibition of the PI3K/AKT pathway and reactivation of FOXOs. This enhanced understanding of TKI activity and induced progenitor cell quiescence suggests that new therapeutic strategies for CML should focus on manipulation of this signaling network.

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.

Targeting hedgehog in hematologic malignancy.

The Hedgehog pathway is a critical mediator of embryonic patterning and organ development, including hematopoiesis. It influences stem cell fate, differentiation, proliferation, and apoptosis in responsive tissues. In adult organisms, hedgehog pathway activity is required for aspects of tissue maintenance and regeneration; however, there is increasing awareness that abnormal hedgehog signaling is associated with malignancy. Hedgehog signaling is critical for early hematopoietic development, but there is controversy over its role in normal hematopoiesis in adult organisms where it may be dispensable. Conversely, hedgehog signaling appears to be an important survival and proliferation signal for a spectrum of hematologic malignancies. Furthermore, hedgehog signaling may be critical for the maintenance and expansion of leukemic stem cells and therefore provides a possible mechanism to selectively target these primitive cell subpopulations, which are resistant to conventional chemotherapy. Indeed, phase 1 clinical trials of hedgehog pathway inhibitors are currently underway to test this hypothesis in myeloid leukemias. This review covers: (1) the hedgehog pathway and its role in normal and malignant hematopoiesis, (2) the recent development of clinical grade small molecule inhibitors of the pathway, and (3) the potential utility of hedgehog pathway inhibition as a therapeutic strategy in hemato-oncology.

Inhibition of mitochondrial folate metabolism drives differentiation through mTORC1 mediated purine sensing.

Supporting cell proliferation through nucleotide biosynthesis is an essential requirement for cancer cells. Hence, inhibition of folate-mediated one carbon (1C) metabolism, which is required for nucleotide synthesis, has been successfully exploited in anti-cancer therapy. Here, we reveal that mitochondrial folate metabolism is upregulated in patient-derived leukaemic stem cells (LSCs). We demonstrate that inhibition of mitochondrial 1C metabolism through impairment of de novo purine synthesis has a cytostatic effect on chronic myeloid leukaemia (CML) cells. Consequently, changes in purine nucleotide levels lead to activation of AMPK signalling and suppression of mTORC1 activity. Notably, suppression of mitochondrial 1C metabolism increases expression of erythroid differentiation markers. Moreover, we find that increased differentiation occurs independently of AMPK signalling and can be reversed through reconstitution of purine levels and reactivation of mTORC1. Of clinical relevance, we identify that combination of 1C metabolism inhibition with imatinib, a frontline treatment for CML patients, decreases the number of therapy-resistant CML LSCs in a patient-derived xenograft model. Our results highlight a role for folate metabolism and purine sensing in stem cell fate decisions and leukaemogenesis.