Lifting the Differentiation Embargo.

Effective differentiation therapy for acute myeloid leukemia (AML) has been restricted to a small subset of patients with one defined genetic abnormality. Using an unbiased small molecule screen, Sykes et al. now identify a mechanism of de-repression of differentiation in several models of AML driven by distinct genetic drivers.

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.

hsa-mir183/EGR1-mediated regulation of E2F1 is required for CML stem/progenitor cell survival.

Chronic myeloid leukemia (CML) stem/progenitor cells (SPCs) express a transcriptional program characteristic of proliferation, yet can achieve and maintain quiescence. Understanding the mechanisms by which leukemic SPCs maintain quiescence will help to clarify how they persist during long-term targeted treatment. We have identified a novel BCR-ABL1 protein kinase-dependent pathway mediated by the upregulation of hsa-mir183, the downregulation of its direct target early growth response 1 (EGR1), and, as a consequence, upregulation of E2F1. We show here that inhibition of hsa-mir183 reduced proliferation and impaired colony formation of CML SPCs. Downstream of this, inhibition of E2F1 also reduced proliferation of CML SPCs, leading to p53-mediated apoptosis. In addition, we demonstrate that E2F1 plays a pivotal role in regulating CML SPC proliferation status. Thus, for the first time, we highlight the mechanism of hsa-mir183/EGR1-mediated E2F1 regulation and demonstrate this axis as a novel, critical factor for CML SPC survival, offering new insights into leukemic stem cell eradication.

The chronic myeloid leukemia stem cell: stemming the tide of persistence.

Chronic myeloid leukemia (CML) is caused by the acquisition of the tyrosine kinase BCR-ABL1 in a hemopoietic stem cell, transforming it into a leukemic stem cell (LSC) that self-renews, proliferates, and differentiates to give rise to a myeloproliferative disease. Although tyrosine kinase inhibitors (TKIs) that target the kinase activity of BCR-ABL1 have transformed CML from a once-fatal disease to a manageable one for the vast majority of patients, only ∼10% of those who present in chronic phase (CP) can discontinue TKI treatment and maintain a therapy-free remission. Strong evidence now shows that CML LSCs are resistant to the effects of TKIs and persist in all patients on long-term therapy, where they may promote acquired TKI resistance, drive relapse or disease progression, and inevitably represent a bottleneck to cure. Since their discovery in patients almost 2 decades ago, CML LSCs have become a well-recognized exemplar of the cancer stem cell and have been characterized extensively, with the aim of developing new curative therapeutic approaches based on LSC eradication. This review summarizes our current understanding of many of the pathways and mechanisms that promote the survival of the CP CML LSCs and how they can be a source of new gene coding mutations that impact in the clinic. We also review recent preclinical approaches that show promise to eradicate the LSC, and future challenges on the path to cure.

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.

Do we need more drugs for chronic myeloid leukemia?

The introduction of protein tyrosine kinase inhibitors (TKIs) in 1998 transformed the management of chronic myeloid leukemia (CML), leading to significantly reduced mortality and improved 5 year survival rates. However, the CML community is faced with several clinical issues that need to be addressed. Ten to 15% of CML patients are diagnosed in advanced phase, and small numbers of chronic phase (CP) cases experience disease progression each year during treatment. For these patients, TKIs induce only transient responses and alternative treatment strategies are urgently required. Depending on choice of first line TKI, approximately 30% of CML CP cases show suboptimal responses, due to a combination of poor compliance, drug intolerance, and drug resistance, with approximately 50% of TKI-resistance caused by kinase domain mutations and the remainder due to unknown mechanisms. Finally, the chance of successful treatment discontinuation is on the order of only 10-20% related to disease persistence. Disease persistence is a poorly understood phenomenon; all CML patients have functional Philadelphia positive (Ph+) stem and progenitor cells in their bone marrows and continue to express BCR-ABL1 by DNA PCR, even when in very deep remission and following treatment discontinuation. What controls the maintenance of these persisting cells, whether it is necessary to fully eradicate the malignant clone to achieve cure, and how that might be approached therapeutically are open questions.

Inhibition of interleukin-1 signaling enhances elimination of tyrosine kinase inhibitor-treated CML stem cells.

Treatment of chronic myelogenous leukemia (CML) with BCR-ABL tyrosine kinase inhibitors (TKI) fails to eliminate leukemia stem cells (LSC). Patients remain at risk for relapse, and additional approaches to deplete CML LSC are needed to enhance the possibility of discontinuing TKI treatment. We have previously reported that expression of the pivotal proinflammatory cytokine interleukin-1 (IL-1) is increased in CML bone marrow. We show here that CML LSC demonstrated increased expression of the IL-1 receptors, IL-1 receptor accessory protein and IL-1 receptor type 1 (IL-1R1), and enhanced sensitivity to IL-1-induced NF-κB signaling compared with normal stem cells. Treatment with recombinant IL-1 receptor antagonist (IL-1RA) inhibited IL-1 signaling in CML LSC and inhibited growth of CML LSC. Importantly, the combination of IL-1RA with TKI resulted in significantly greater inhibition of CML LSC compared with TKI alone. Our studies also suggest that IL-1 signaling contributes to overexpression of inflammatory mediators in CML LSC, suggesting that blocking IL-1 signaling could modulate the inflammatory milieu. We conclude that IL-1 signaling contributes to maintenance of CML LSC following TKI treatment and that IL-1 blockade with IL-1RA enhances elimination of TKI-treated CML LSC. These results provide a strong rationale for further exploration of anti-IL-1 strategies to enhance LSC elimination in CML.

Adult hematopoietic stem cells lacking Hif-1α self-renew normally.

The hematopoietic stem cell (HSC) pool is maintained under hypoxic conditions within the bone marrow microenvironment. Cellular responses to hypoxia are largely mediated by the hypoxia-inducible factors, Hif-1 and Hif-2. The oxygen-regulated α subunits of Hif-1 and Hif-2 (namely, Hif-1α and Hif-2α) form dimers with their stably expressed ß subunits and control the transcription of downstream hypoxia-responsive genes to facilitate adaptation to low oxygen tension. An initial study concluded that Hif-1α is essential for HSC maintenance, whereby Hif-1α-deficient HSCs lost their ability to self-renew in serial transplantation assays. In another study, we demonstrated that Hif-2α is dispensable for cell-autonomous HSC maintenance, both under steady-state conditions and following transplantation. Given these unexpected findings, we set out to revisit the role of Hif-1α in cell-autonomous HSC functions. Here we demonstrate that inducible acute deletion of Hif-1α has no impact on HSC survival. Notably, unstressed HSCs lacking Hif-1α efficiently self-renew and sustain long-term multilineage hematopoiesis upon serial transplantation. Finally, Hif-1α-deficient HSCs recover normally after hematopoietic injury induced by serial administration of 5-fluorouracil. We therefore conclude that despite the hypoxic nature of the bone marrow microenvironment, Hif-1α is dispensable for cell-autonomous HSC maintenance.

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.

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.

Dipeptidylpeptidase IV (CD26) defines leukemic stem cells (LSC) in chronic myeloid leukemia.

Chronic myeloid leukemia (CML) is a stem cell (SC) neoplasm characterized by the BCR/ABL1 oncogene. Although mechanisms of BCR/ABL1-induced transformation are well-defined, little is known about effector-molecules contributing to malignant expansion and the extramedullary spread of leukemic SC (LSC) in CML. We have identified the cytokine-targeting surface enzyme dipeptidylpeptidase-IV (DPPIV/CD26) as a novel, specific and pathogenetically relevant biomarker of CD34(+)/CD38(─) CML LSC. In functional assays, CD26 was identified as target enzyme disrupting the SDF-1-CXCR4-axis by cleaving SDF-1, a chemotaxin recruiting CXCR4(+) SC. CD26 was not detected on normal SC or LSC in other hematopoietic malignancies. Correspondingly, CD26(+) LSC decreased to low or undetectable levels during successful treatment with imatinib. CD26(+) CML LSC engrafted NOD-SCID-IL-2Rγ(-/-) (NSG) mice with BCR/ABL1(+) cells, whereas CD26(─) SC from the same patients produced multilineage BCR/ABL1(-) engraftment. Finally, targeting of CD26 by gliptins suppressed the expansion of BCR/ABL1(+) cells. Together, CD26 is a new biomarker and target of CML LSC. CD26 expression may explain the abnormal extramedullary spread of CML LSC, and inhibition of CD26 may revert abnormal LSC function and support curative treatment approaches in this malignancy.

Redirecting traffic using the XPO1 police.

In this issue of Blood, Walker et al investigate the preclinical potential of KPT-330, an exportin-1 (XPO1, also known as chromosome maintenance protein 1 [CRM1]) inhibitor, against both accelerated phase (AP) and blast crisis chronic myeloid leukemia (CML-BC) and against Philadelphia chromosome-positive (Ph1) acute lymphoblastic leukemia (ALL), all of which are diseases of significant unmet clinical need.1 The authors provide encouraging data from both a leukemic mouse model and a single CML-AP patient, corroborating mechanistic studies suggesting that KPT-330 efficacy relies on targeting abundantly expressed XPO1, followed by the reactivation of protein phosphatase 2A (PP2A).

Microenvironmental protection of CML stem and progenitor cells from tyrosine kinase inhibitors through N-cadherin and Wnt-ß-catenin signaling.

Tyrosine kinase inhibitors (TKIs) are highly effective in treatment of chronic myeloid leukemia (CML) but do not eliminate leukemia stem cells (LSCs), which remain a potential source of relapse. TKI treatment effectively inhibits BCR-ABL kinase activity in CML LSCs, suggesting that additional kinase-independent mechanisms contribute to LSC preservation. We investigated whether signals from the bone marrow (BM) microenvironment protect CML LSCs from TKI treatment. Coculture with human BM mesenchymal stromal cells (MSCs) significantly inhibited apoptosis and preserved CML stem/progenitor cells following TKI exposure, maintaining colony-forming ability and engraftment potential in immunodeficient mice. We found that the N-cadherin receptor plays an important role in MSC-mediated protection of CML progenitors from TKI. N-cadherin-mediated adhesion to MSCs was associated with increased cytoplasmic N-cadherin-ß-catenin complex formation as well as enhanced ß-catenin nuclear translocation and transcriptional activity. Increased exogenous Wnt-mediated ß-catenin signaling played an important role in MSC-mediated protection of CML progenitors from TKI treatment. Our results reveal a close interplay between N-cadherin and the Wnt-ß-catenin pathway in protecting CML LSCs during TKI treatment. Importantly, these results reveal novel mechanisms of resistance of CML LSCs to TKI treatment and suggest new targets for treatment designed to eradicate residual LSCs in CML patients.

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.

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.

The hOCT1 SNPs M420del and M408V alter imatinib uptake and M420del modifies clinical outcome in imatinib-treated chronic myeloid leukemia.

Although the prognosis of chronic myeloid leukemia (CML) patients treated with imatinib is good, many fail to develop an optimal response or lose one. This heterogeneity could be attributed to the presence of human organic cation transporter-1 (hOCT1) single nucleotide polymorphisms (SNPs). In the present study, we analyzed the effect of 23 hOCT1 SNPs on imatinib treatment outcome in newly diagnosed CML patients using MassARRAY sequencing and pyrosequencing. The only SNP associated with outcome was M420del (rs35191146), with patients with the M420del demonstrating an increased probability of imatinib treatment failure. In CML cell lines transfected with M420del and/or M408V, M420del significantly decreased imatinib uptake, but this effect was countered if the M408V (rs628031) SNP was also present. A similar effect was seen for the uptake of the hOCT1 substrates TEA(+) and ASP(+). Finally, apparent hOCT1 mRNA levels were studied using both our earlier primers covering the M420del and another set that did not. Different mRNA expression was observed, explaining the disparity in published data on the prognostic importance of hOCT1 mRNA and highlighting the importance of avoiding common SNP sites in primer design. These data demonstrate that the common M420del SNP can modulate the outcome of imatinib treatment.

Genomic instability may originate from imatinib-refractory chronic myeloid leukemia stem cells.

Genomic instability is a hallmark of chronic myeloid leukemia in chronic phase (CML-CP) resulting in BCR-ABL1 mutations encoding resistance to tyrosine kinase inhibitors (TKIs) and/or additional chromosomal aberrations leading to disease relapse and/or malignant progression. TKI-naive and TKI-treated leukemia stem cells (LSCs) and leukemia progenitor cells (LPCs) accumulate high levels of reactive oxygen species (ROS) and oxidative DNA damage. To determine the role of TKI-refractory LSCs in genomic instability, we used a murine model of CML-CP where ROS-induced oxidative DNA damage was elevated in LSCs, including quiescent LSCs, but not in LPCs. ROS-induced oxidative DNA damage in LSCs caused clinically relevant genomic instability in CML-CP-like mice, such as TKI-resistant BCR-ABL1 mutations (E255K, T315I, H396P), deletions in Ikzf1 and Trp53, and additions in Zfp423 and Idh1. Despite inhibition of BCR-ABL1 kinase, imatinib did not downregulate ROS and oxidative DNA damage in TKI-refractory LSCs to the levels detected in normal cells, and CML-CP-like mice treated with imatinib continued to accumulate clinically relevant genetic aberrations. Inhibition of class I p21-activated protein kinases by IPA3 downregulated ROS in TKI-naive and TKI-treated LSCs. Altogether, we postulate that genomic instability may originate in the most primitive TKI-refractory LSCs in TKI-naive and TKI-treated patients.

Hif-2α is not essential for cell-autonomous hematopoietic stem cell maintenance.

Local hypoxia in hematopoietic stem cell (HSC) niches is thought to regulate HSC functions. Hypoxia-inducible factor-1 (Hif-1) and Hif-2 are key mediators of cellular responses to hypoxia. Although oxygen-regulated α-subunits of Hifs, namely Hif-1α and Hif-2α, are closely related, they play overlapping and also distinct functions in nonhematopoietic tissues. Although Hif-1α-deficient HSCs lose their activity on serial transplantation, the role for Hif-2α in cell-autonomous HSC maintenance remains unknown. Here, we demonstrate that constitutive or inducible hematopoiesis-specific Hif-2α deletion does not affect HSC numbers and steady-state hematopoiesis. Furthermore, using serial transplantations and 5-fluorouracil treatment, we demonstrate that HSCs do not require Hif-2α to self-renew and recover after hematopoietic injury. Finally, we show that Hif-1α deletion has no major impact on steady-state maintenance of Hif-2α-deficient HSCs and their ability to repopulate primary recipients, indicating that Hif-1α expression does not account for normal behavior of Hif-2α-deficient HSCs.

Concise review: cancer cells escape from oncogene addiction: understanding the mechanisms behind treatment failure for more effective targeting.

Oncogene addiction describes the dependence of some cancers on one or a few genes for their survival. Inhibition of the corresponding oncoproteins can lead to dramatic responses. However, in some cases, such as chronic myeloid leukemia (CML), a disease characterized by the presence of the abnormal fusion tyrosine kinase BCR-ABL, cancer stem cells may never acquire addiction to the oncogene that drives disease development. The suggested mechanism(s) for treatment failure include a quiescent stem cell population capable of reinstating disease, high levels of oncoprotein expression, or acquired mutations in the oncogene. In this review, we discuss the evidence for oncogene addiction in several solid tumors and their potential escape mechanism(s) with a particular focus on CML stem cells.