Advertisement

A rare subgroup of leukemia stem cells harbors relapse-inducing potential in acute lymphoblastic leukemia

  • Daniela Senft
    Affiliations
    Research Unit Apoptosis in Hematopoietic Stem Cells, Helmholtz Center Munich, German Center for Environmental Health (HMGU), Munich, Germany
    Search for articles by this author
  • Irmela Jeremias
    Correspondence
    Offprint requests to: Irmela Jeremias, Helmholtz Center Munich, Marchioninistraße 25, 81377 Munich, Germany
    Affiliations
    Research Unit Apoptosis in Hematopoietic Stem Cells, Helmholtz Center Munich, German Center for Environmental Health (HMGU), Munich, Germany

    Department of Pediatrics, Dr. von Hauner Childrens Hospital, Ludwig Maximilians University, Munich, Germany

    German Consortium for Translational Cancer Research (DKTK), Partnering Site Munich, Munich, Germany
    Search for articles by this author
Open AccessPublished:September 24, 2018DOI:https://doi.org/10.1016/j.exphem.2018.09.006
      After initially successful chemotherapy, relapse frequently jeopardizes the outcome of patients with acute leukemia. Because of their adverse characteristics of self-renewal and dormancy, leukemia stem cells have been hypothesized to play a critical role in resistance to antiproliferative chemotherapy and the development of relapse. The high abundance of stem-like cells in acute lymphoblastic leukemia (ALL), however, suggests that not all leukemia-initiating cells carry these adverse characteristics, complicating the biological characterization of relapse-inducing cells in this malignancy. Here, we review sources of therapy resistance and relapse in acute leukemias, which include tumor cell plasticity and reversible characteristics. We discuss the development of patient-derived mouse models that are genetically engineered to mimic long-term dormancy and minimal residual disease in patients. These models allow the tracking and functional characterization of patient-derived ALL blasts that combine the properties of long-term dormancy, treatment resistance, and stemness. Finally, we discuss possible therapeutic avenues to target the functional plasticity of leukemia-initiating cells in ALL.

      Cancer stem cells as a source for therapy resistance and relapse

      Acute leukemias of lymphoid (acute lymphoblastic leukemia; ALL) or myeloid (acute myeloid leukemia; AML) lineage arise from hematopoietic stem or progenitor cells in the bone marrow. Patients with acute leukemia often achieve complete remission with induction chemotherapy, yet many will experience relapse, which poses a major obstacle to cure [
      • Inaba H
      • Greaves M
      • Mullighan CG
      Acute lymphoblastic leukaemia.
      ,
      • Dohner H
      • Weisdorf DJ
      • Bloomfield CD
      Acute myeloid leukemia.
      ]. Leukemia relapse has been suggested to arise from a small subpopulation of cells that survive chemotherapy, persist as minimal residual disease (MRD) in complete remission, and ultimately reinitiate malignant growth. Although MRD detection in acute leukemia has a strong prognostic value [
      • Inaba H
      • Greaves M
      • Mullighan CG
      Acute lymphoblastic leukaemia.
      ,
      • Dohner H
      • Weisdorf DJ
      • Bloomfield CD
      Acute myeloid leukemia.
      ], MRD is not an absolute marker of relapse because not all residual cells may have the functional capability to proliferate into relapse [
      • Luskin MR
      • Murakami MA
      • Manalis SR
      • Weinstock DM
      Targeting minimal residual disease. a path to cure?.
      ]. Accordingly, relapse-inducing cells are a “subpopulation” of MRD cells with important functional properties, including the ability to repopulate the tumor. One possible explanation for this clinical behaviour can be provided by the cancer stem cell model, which is based on the idea that cancers, like normal tissues, are maintained by a rare, biologically distinct subpopulation of cells that have the capacity for long-term tumor propagation and self-renewal and give rise to progeny that lack these characteristics. Such cancer stem cells are thought to be highly resistant to therapeutic regimens, survive chemotherapy, and ultimately lead to relapse [
      • Kreso A
      • Dick JE
      Evolution of the cancer stem cell model.
      ,
      • Batlle E
      • Clevers H
      Cancer stem cells revisited.
      ].
      The conceptual framework of a leukemia stem cell model is based on a putative analogy between normal and malignant hematopoiesis. Normal hematopoiesis is maintained by hematopoietic stem cells (HSCs), a rare subpopulation of bone marrow cells with multilineage differentiation and self-renewal capacity. HSCs give rise to highly proliferating hematopoietic progenitor cells, which generate all mature blood cells [
      • Laurenti E
      • Gottgens B
      From haematopoietic stem cells to complex differentiation landscapes.
      ]. Different cell types within the bone marrow, including mesenchymal stem cells (MSCs, cells that form adipocytes, osteoblasts, and chondrocytes), endothelial cells, and nerve fibers, form the stem cell niche, a specialized microenvironment that allows HSC maintenance [
      • Crane GM
      • Jeffery E
      • Morrison SJ
      Adult haematopoietic stem cell niches.
      ,
      • Trumpp A
      • Essers M
      • Wilson A
      Awakening dormant haematopoietic stem cells.
      ]. At the top of the hematopoietic hierarchy are extremely slow cycling, long-term dormant HSCs that carry the highest reconstitution potential [
      • Wilson A
      • Laurenti E
      • Oser G
      • et al.
      Hematopoietic stem cells reversibly switch from dormancy to self–renewal during homeostasis and repair.
      ,
      • Foudi A
      • Hochedlinger K
      • Van Buren D
      • et al.
      Analysis of histone 2B-GFP retention reveals slowly cycling hematopoietic stem cells.
      ,
      • Qiu J
      • Papatsenko D
      • Niu X
      • Schaniel C
      • Moore K
      Divisional history and hematopoietic stem cell function during homeostasis.
      ,
      • Bernitz JM
      • Kim HS
      • MacArthur B
      • Sieburg H
      • Moore K
      Hematopoietic stem cells count and remember self-renewal divisions.
      ,
      • Cabezas-Wallscheid N
      • Buettner F
      • Sommerkamp P
      • et al.
      Vitamin A-retinoic acid signaling regulates hematopoietic stem cell dormancy.
      ,
      • Takizawa H
      • Regoes RR
      • Boddupalli CS
      • Bonhoeffer S
      • Manz MG
      Dynamic variation in cycling of hematopoietic stem cells in steady state and inflammation.
      ]. Their contribution to steady-state blood formation may be limited because it is mediated by more actively cycling HSCs with restricted long-term propagation potential. The dormant HSC subsets can readily be activated upon stress, for example, by interferons, lipopolysaccharides, or chemotherapy among others, and significantly contribute to repair processes [
      • Trumpp A
      • Essers M
      • Wilson A
      Awakening dormant haematopoietic stem cells.
      ,
      • Boettcher S
      • Manz MG
      Regulation of inflammation- and infection-driven hematopoiesis.
      ]. Importantly, the transition from dormant to active HSCs is a reversible process and HSCs can return into a deep quiescent state following stress [
      • Wilson A
      • Laurenti E
      • Oser G
      • et al.
      Hematopoietic stem cells reversibly switch from dormancy to self–renewal during homeostasis and repair.
      ,
      • Takizawa H
      • Regoes RR
      • Boddupalli CS
      • Bonhoeffer S
      • Manz MG
      Dynamic variation in cycling of hematopoietic stem cells in steady state and inflammation.
      ], at least for a limited number of cell divisions [
      • Bernitz JM
      • Kim HS
      • MacArthur B
      • Sieburg H
      • Moore K
      Hematopoietic stem cells count and remember self-renewal divisions.
      ]. Dormancy of adult stem cells has been suggested as a means to survive chemotherapeutic and other DNA-damaging challenges [
      • Wilson A
      • Laurenti E
      • Oser G
      • et al.
      Hematopoietic stem cells reversibly switch from dormancy to self–renewal during homeostasis and repair.
      ,
      • Randall TD
      • Weissman IL
      Phenotypic and functional changes induced at the clonal level in hematopoietic stem cells after 5-fluorouracil treatment.
      ], limit the accumulation of mutations, and thus serve as a reserve stem cell pool to maintain normal hematopoiesis following stress [
      • Trumpp A
      • Essers M
      • Wilson A
      Awakening dormant haematopoietic stem cells.
      ].
      Xenotransplantation of AML cells in immunodeficient mice has significantly contributed to the formal verification of a hierarchical organization of cancer because these studies demonstrated that rare stem-like cells with self-renewal capacity sustain AML. Such stem-like cells are termed leukemia-initiating cells (LICs) and they are enriched within the CD34+CD38 immunophenotype[
      • Lapidot T
      • Sirard C
      • Vormoor J
      • et al.
      A cell initiating human acute myeloid leukaemia after transplantation into SCID mice.
      ,
      • Bonnet D
      • Dick JE
      Human acute myeloid leukemia is organized as a hierarchy that originates from a primitive hematopoietic cell.
      ,
      • Dick JE
      Stem cell concepts renew cancer research.
      ]. Although more sophisticated mouse models demonstrated that other fractions (CD34+CD38+ and also CD34) can also contain LICs in AML patient samples, it is now widely accepted that AML and numerous other malignancies are hierarchically organized [
      • Kreso A
      • Dick JE
      Evolution of the cancer stem cell model.
      ]. In analogy to HSCs, AML-LICs are functionally heterogeneous in terms of self-renewal potential [
      • Hope KJ
      • Jin L
      • Dick JE
      Acute myeloid leukemia originates from a hierarchy of leukemic stem cell classes that differ in self-renewal capacity.
      ] and heterogeneous in terms of growth kinetics; at least a subset of LICs is maintained in a quiescent state [
      • Guan Y
      • Gerhard B
      • Hogge DE
      Detection, isolation, and stimulation of quiescent primitive leukemic progenitor cells from patients with acute myeloid leukemia (AML).
      ]. Of therapeutic relevance is the fact that AML-LICs display a similar transcriptional profile as HSCs, which is a powerful indicator of resistance to standard therapy [
      • Ng SW
      • Mitchell A
      • Kennedy JA
      • et al.
      A 17-gene stemness score for rapid determination of risk in acute leukaemia.
      ]. Furthermore, AML-LIC frequency at diagnosis is indicative of a poor outcome [
      • van Rhenen A
      • Feller N
      • Kelder A
      • et al.
      High stem cell frequency in acute myeloid leukemia at diagnosis predicts high minimal residual disease and poor survival.
      ] and frequency of LICs also increases following therapy, providing an explanation for the notorious drug resistance of relapsed AML [
      • Ho TC
      • LaMere M
      • Stevens BM
      • et al.
      Evolution of acute myelogenous leukemia stem cell properties after treatment and progression.
      ]. In agreement, experiments in preclinical models strongly support the notion that AML-LICs are the source of relapse in AML [
      • Fong CY
      • Gilan O
      • Lam EY
      • et al.
      BET inhibitor resistance emerges from leukaemia stem cells.
      ,
      • Shlush LI
      • Mitchell A
      • Heisler L
      • et al.
      Tracing the origins of relapse in acute myeloid leukaemia to stem cells.
      ]. In particular, the quiescence of AML-LICs has been associated with resistance to anti-proliferative chemotherapy [
      • Ishikawa F
      • Yoshida S
      • Saito Y
      • et al.
      Chemotherapy-resistant human AML stem cells home to and engraft within the bone-marrow endosteal region.
      ,
      • Saito Y
      • Uchida N
      • Tanaka S
      • et al.
      Induction of cell cycle entry eliminates human leukemia stem cells in a mouse model of AML.
      ,
      • Essers MA
      • Trumpp A
      Targeting leukemic stem cells by breaking their dormancy.
      ]. Therefore, efforts to break the dormancy of these cells in order to sensitize them to anti-proliferative chemotherapy were suggested as a valuable means to eradicate LICs and achieve long-term remissions [
      • Saito Y
      • Uchida N
      • Tanaka S
      • et al.
      Induction of cell cycle entry eliminates human leukemia stem cells in a mouse model of AML.
      ,
      • Essers MA
      • Trumpp A
      Targeting leukemic stem cells by breaking their dormancy.
      ].
      Although various similarities in the functional behavior of AML-LICs and HSCs have been established, these results are not readily transferable to all hematological malignancies. In ALL, no markers could be identified to prospectively isolate LICs and a high proportion of cells among various immunophenotypes display leukemia-initiating potential in xenotransplantation experiments [
      • Kong Y
      • Yoshida S
      • Saito Y
      • et al.
      CD34+CD38+CD19+ as well as CD34+CD38–CD19+ cells are leukemia-initiating cells with self-renewal capacity in human B-precursor ALL.
      ,
      • le Viseur C
      • Hotfilder M
      • Bomken S
      • et al.
      In childhood acute lymphoblastic leukemia, blasts at different stages of immunophenotypic maturation have stem cell properties.
      ,
      • Rehe K
      • Wilson K
      • Bomken S
      • et al.
      Acute B lymphoblastic leukaemia-propagating cells are present at high frequency in diverse lymphoblast populations.
      ,
      • Elder A
      • Bomken S
      • Wilson I
      • et al.
      Abundant and equipotent founder cells establish and maintain acute lymphoblastic leukaemia.
      ]. Therefore, the hierarchical stem cell model of AML does not apply to ALL, in which tumor progression/relapse may be a stochastic process. Although highly similar regarding the clinical course of treatment response, MRD, and relapse, the two sister malignancies ALL and AML show major differences concerning LIC biology.

      Sources for therapy resistance and relapse in ALL

      Because stemness is an insufficient criterion to define the subpopulation of relapse-inducing cells in ALL, the basic biological conditions that determine relapse remain to be defined.
      Genomic and functional analysis of ALL patient samples revealed a branching evolutionary pattern and multiple subclones coexist at the time of diagnosis and show functional diversity in terms of leukemia-initiating potential [
      • Anderson K
      • Lutz C
      • van Delft FW
      • et al.
      Genetic variegation of clonal architecture and propagating cells in leukaemia.
      ,
      • Bardini M
      • Woll PS
      • Corral L
      • et al.
      Clonal variegation and dynamic competition of leukemia–initiating cells in infant acute lymphoblastic leukemia with MLL rearrangement.
      ,
      • Notta F
      • Mullighan CG
      • Wang JC
      • et al.
      Evolution of human BCR-ABL1 lymphoblastic leukaemia–initiating cells.
      ]. Such genetic diversity can provide a basis for the clonal selection of chemoresistant clones by therapeutic regimens. In agreement, genomic studies of paired diagnosis and relapse samples demonstrated that, at relapse, ALL blasts are derived from minor subclones present at diagnosis or evolve from ancestral diagnostic clones, but rarely from the main diagnostic clone [
      • Ding LW
      • Sun QY
      • Tan KT
      • et al.
      Mutational landscape of pediatric acute lymphoblastic leukemia.
      ,
      • Ma X
      • Edmonson M
      • Yergeau D
      • et al.
      Rise and fall of subclones from diagnosis to relapse in pediatric B–acute lymphoblastic leukaemia.
      ,
      • Mullighan CG
      • Phillips LA
      • Su X
      • et al.
      Genomic analysis of the clonal origins of relapsed acute lymphoblastic leukemia.
      ,
      • Oshima K
      • Khiabanian H
      • da Silva-Almeida AC
      • et al.
      Mutational landscape, clonal evolution patterns, and role of RAS mutations in relapsed acute lymphoblastic leukemia.
      ]. Multiple genetic variants that confer resistance to standard therapy are enriched in relapsed ALL samples. These include NT5C2 [
      • Meyer JA
      • Wang J
      • Hogan LE
      • et al.
      Relapse-specific mutations in NT5C2 in childhood acute lymphoblastic leukemia.
      ,
      • Tzoneva G
      • Perez-Garcia A
      • Carpenter Z
      • et al.
      Activating mutations in the NT5C2 nucleotidase gene drive chemotherapy resistance in relapsed ALL.
      ] and PRPS1 [
      • Li B
      • Li H
      • Bai Y
      • et al.
      Negative feedback-defective PRPS1 mutants drive thiopurine resistance in relapsed childhood ALL.
      ], which mediate resistance to genotoxic agents; Alterations in CREBBP [
      • Mullighan CG
      • Zhang J
      • Kasper LH
      • et al.
      CREBBP mutations in relapsed acute lymphoblastic leukaemia.
      ] contribute to glucocorticoid resistance and CDKN2A/B [
      • Inaba H
      • Greaves M
      • Mullighan CG
      Acute lymphoblastic leukaemia.
      ,
      • Notta F
      • Mullighan CG
      • Wang JC
      • et al.
      Evolution of human BCR-ABL1 lymphoblastic leukaemia–initiating cells.
      ,
      • Mullighan CG
      • Phillips LA
      • Su X
      • et al.
      Genomic analysis of the clonal origins of relapsed acute lymphoblastic leukemia.
      ] or RAS pathway [
      • Oshima K
      • Khiabanian H
      • da Silva-Almeida AC
      • et al.
      Mutational landscape, clonal evolution patterns, and role of RAS mutations in relapsed acute lymphoblastic leukemia.
      ,
      • Irving J
      • Matheson E
      • Minto L
      • et al.
      Ras pathway mutations are prevalent in relapsed childhood acute lymphoblastic leukemia and confer sensitivity to MEK inhibition.
      ] alterations that are associated with resistance to multiple drugs. However, sequence analysis alone may not reveal all features of relapse-inducing cells in ALL. For example, although RAS pathway mutations are associated with poor prognosis [
      • Oshima K
      • Khiabanian H
      • da Silva-Almeida AC
      • et al.
      Mutational landscape, clonal evolution patterns, and role of RAS mutations in relapsed acute lymphoblastic leukemia.
      ,
      • Irving J
      • Matheson E
      • Minto L
      • et al.
      Ras pathway mutations are prevalent in relapsed childhood acute lymphoblastic leukemia and confer sensitivity to MEK inhibition.
      ], their presence at diagnosis does not strictly predict relapse; some mutations in this pathway can be lost at relapse or are present in diagnostic samples that never relapse [
      • Ding LW
      • Sun QY
      • Tan KT
      • et al.
      Mutational landscape of pediatric acute lymphoblastic leukemia.
      ,
      • Irving J
      • Matheson E
      • Minto L
      • et al.
      Ras pathway mutations are prevalent in relapsed childhood acute lymphoblastic leukemia and confer sensitivity to MEK inhibition.
      ,
      • Molteni CG
      • Te Kronnie G
      • Bicciato S
      • et al.
      PTPN11 mutations in childhood acute lymphoblastic leukemia occur as a secondary event associated with high hyperdiploidy.
      ]. This, together with the facts that pediatric and haematological cancers have the lowest somatic mutation rate and most cases of ALL are genetically stable [
      • Grobner SN
      • Worst BC
      • Weischenfeldt J
      • et al.
      The landscape of genomic alterations across childhood cancers.
      ,
      • Ma X
      • Liu Y
      • Liu Y
      • et al.
      Pan-cancer genome and transcriptome analyses of 1,699 paediatric leukaemias and solid tumours.
      ] and that not all relapses are derived from minority subclones but can sometimes derive from the major diagnostic clone [
      • Ding LW
      • Sun QY
      • Tan KT
      • et al.
      Mutational landscape of pediatric acute lymphoblastic leukemia.
      ,
      • Ma X
      • Edmonson M
      • Yergeau D
      • et al.
      Rise and fall of subclones from diagnosis to relapse in pediatric B–acute lymphoblastic leukaemia.
      ,
      • Mullighan CG
      • Phillips LA
      • Su X
      • et al.
      Genomic analysis of the clonal origins of relapsed acute lymphoblastic leukemia.
      ,
      • Oshima K
      • Khiabanian H
      • da Silva-Almeida AC
      • et al.
      Mutational landscape, clonal evolution patterns, and role of RAS mutations in relapsed acute lymphoblastic leukemia.
      ,
      • Spinella JF
      • Richer C
      • Cassart P
      • Ouimet M
      • Healy J
      • Sinnett D
      Mutational dynamics of early and late relapsed childhood ALL: Rapid clonal expansion and long-term dormancy.
      ,
      • Kunz JB
      • Rausch T
      • Bandapalli OR
      • et al.
      Pediatric T-cell lymphoblastic leukemia evolves into relapse by clonal selection, acquisition of mutations and promoter hypomethylation.
      ], strongly suggests that nongenetic factors significantly contribute to relapse. Although hardwired genetic mechanisms may account for refractory disease and early relapse, different mechanisms may account for late relapses that can occur even decades after the initial diagnosis [
      • Spinella JF
      • Richer C
      • Cassart P
      • Ouimet M
      • Healy J
      • Sinnett D
      Mutational dynamics of early and late relapsed childhood ALL: Rapid clonal expansion and long-term dormancy.
      ,
      • Ford AM
      • Mansur MB
      • Furness CL
      • et al.
      Protracted dormancy of pre-leukemic stem cells.
      ]. Therefore, both clonal architecture and stemness remain insufficient criteria to define relapse-inducing cells in ALL.
      Cellular plasticity in epigenetic programs (which is influenced by the genetic background as well as the tumor microenvironment) is considered the underlying cause of functional heterogeneity in cancer [
      • Batlle E
      • Clevers H
      Cancer stem cells revisited.
      ,
      • Meacham CE
      • Morrison SJ
      Tumour heterogeneity and cancer cell plasticity.
      ,
      • Meads MB
      • Gatenby RA
      • Dalton WS
      Environment-mediated drug resistance: A major contributor to minimal residual disease.
      ]. In response to appropriate environmental stimuli, tumor cells may be temporarily endowed with certain traits, including tumor-/leukemia-initiating capacity and therapy resistance, as has been suggested in diverse cancers [
      • Knoechel B
      • Roderick JE
      • Williamson KE
      • et al.
      An epigenetic mechanism of resistance to targeted therapy in T cell acute lymphoblastic leukemia.
      ,
      • Kreso A
      • O'Brien CA
      • van Galen P
      • et al.
      Variable clonal repopulation dynamics influence chemotherapy response in colorectal cancer.
      ,
      • Roesch A
      • Fukunaga-Kalabis M
      • Schmidt EC
      • et al.
      A temporarily distinct subpopulation of slow–cycling melanoma cells is required for continuous tumor growth.
      ,
      • Roesch A
      • Vultur A
      • Bogeski I
      • et al.
      Overcoming intrinsic multidrug resistance in melanoma by blocking the mitochondrial respiratory chain of slow-cycling JARID1B(high) cells.
      ,
      • Sharma SV
      • Lee DY
      • Li B
      • et al.
      A chromatin-mediated reversible drug-tolerant state in cancer cell subpopulations.
      ,
      • Viale A
      • Pettazzoni P
      • Lyssiotis CA
      • et al.
      Oncogene ablation-resistant pancreatic cancer cells depend on mitochondrial function.
      ]. The reversibility of drug resistance, for example, has been demonstrated in various cancer cell lines in response to targeted EGFR or BRAF inhibitors or cisplatin [
      • Sharma SV
      • Lee DY
      • Li B
      • et al.
      A chromatin-mediated reversible drug-tolerant state in cancer cell subpopulations.
      ], as well as in T-cell ALL (T-ALL) xenografts following y-secretase inhibition [
      • Knoechel B
      • Roderick JE
      • Williamson KE
      • et al.
      An epigenetic mechanism of resistance to targeted therapy in T cell acute lymphoblastic leukemia.
      ]. Interestingly, the number of these drug-tolerant persisting cells can be decreased and establishment of resistance prevented in vitro by simultaneous treatment with histone deacetylase (HDAC) inhibitors [
      • Sharma SV
      • Lee DY
      • Li B
      • et al.
      A chromatin-mediated reversible drug-tolerant state in cancer cell subpopulations.
      ]. Similarly, in patient-derived xenografts of pediatric T-ALL, combined inhibition of NOTCH and BRD4, a chromatin regulator upregulated in persistent T-ALL cells, significantly prolonged survival compared with single-agent treatment [
      • Knoechel B
      • Roderick JE
      • Williamson KE
      • et al.
      An epigenetic mechanism of resistance to targeted therapy in T cell acute lymphoblastic leukemia.
      ]. The interaction of tumor cells with the microenvironment has been suggested as a major regulator of such reversible traits [
      • Batlle E
      • Clevers H
      Cancer stem cells revisited.
      ,
      • Meacham CE
      • Morrison SJ
      Tumour heterogeneity and cancer cell plasticity.
      ,
      • Meads MB
      • Gatenby RA
      • Dalton WS
      Environment-mediated drug resistance: A major contributor to minimal residual disease.
      ]. Evidence that environmentally mediated drug resistance may protect ALL blasts from therapy comes from in vitro experiments, wherein the interaction of ALL blasts with stromal cells is essential for their survival in vitro and can protect blasts against asparaginase and prednisolone [
      • Polak R
      • de Rooij B
      • Pieters R
      • den Boer ML
      B-cell precursor acute lymphoblastic leukemia cells use tunneling nanotubes to orchestrate their microenvironment.
      ,
      • Iwamoto S
      • Mihara K
      • Downing JR
      • Pui CH
      • Campana D
      Mesenchymal cells regulate the response of acute lymphoblastic leukemia cells to asparaginase.
      ]. Accordingly, safe-haven niches that protect leukemic blasts from chemotherapy have been identified in the bone marrow in vivo, wherein mesenchymal stem cells [
      • Duan CW
      • Shi J
      • Chen J
      • et al.
      Leukemia-propagating cells rebuild an evolving niche in response to therapy.
      ], osteoblasts [
      • Boyerinas B
      • Zafrir M
      • Yesilkanal AE
      • Price TT
      • Hyjek EM
      • Sipkins DA
      Adhesion to osteopontin in the bone marrow niche regulates lymphoblastic leukemia cell dormancy.
      ], or other stromal cell types in the microenvironment [
      • Cahu X
      • Calvo J
      • Poglio S
      • et al.
      Bone marrow sites differently imprint dormancy and chemoresistance to T-cell acute lymphoblastic leukemia.
      ] protect leukemic blasts from therapy. In response to chemotherapy, ALL blasts can also actively remodel their environment via the recruitment of mesenchymal cells and the establishment of such a protective niche can be found in patient samples of partial responders or nonresponders [
      • Duan CW
      • Shi J
      • Chen J
      • et al.
      Leukemia-propagating cells rebuild an evolving niche in response to therapy.
      ].
      Interestingly, regardless of the model tested, quiescence of cancer cells seems to be a common characteristic of therapy-resistant subpopulations: Quiescent glioblastoma stem cells survive chemotherapy and reinitiate malignant growth following remission [
      • Chen J
      • Li Y
      • Yu TS
      • et al.
      A restricted cell population propagates glioblastoma growth after chemotherapy.
      ] and, in diverse cancer cell lines, quiescent, drug-tolerant cells persist during the course of therapy [
      • Sharma SV
      • Lee DY
      • Li B
      • et al.
      A chromatin-mediated reversible drug-tolerant state in cancer cell subpopulations.
      ]. Additionally, quiescent cells survive oncogene ablation in a mouse model of pancreatic cancer [
      • Viale A
      • Pettazzoni P
      • Lyssiotis CA
      • et al.
      Oncogene ablation-resistant pancreatic cancer cells depend on mitochondrial function.
      ], and in colorectal cancer xenografts, a previously dormant cell population becomes dominant following chemotherapy [
      • Kreso A
      • O'Brien CA
      • van Galen P
      • et al.
      Variable clonal repopulation dynamics influence chemotherapy response in colorectal cancer.
      ]. Similarly, slow-cycling melanoma cells display a general drug-resistant phenotype [
      • Roesch A
      • Fukunaga-Kalabis M
      • Schmidt EC
      • et al.
      A temporarily distinct subpopulation of slow–cycling melanoma cells is required for continuous tumor growth.
      ,
      • Roesch A
      • Vultur A
      • Bogeski I
      • et al.
      Overcoming intrinsic multidrug resistance in melanoma by blocking the mitochondrial respiratory chain of slow-cycling JARID1B(high) cells.
      ] and a subpopulation of slow-cycling transforming growth factor-beta-responsive squamous cell carcinoma stem cells survive chemotherapy and induce relapse in xenotransplantation experiments [
      • Oshimori N
      • Oristian D
      • Fuchs E
      TGF-beta promotes heterogeneity and drug resistance in squamous cell carcinoma.
      ]. These studies suggest that, regardless of the presence or absence of a strict hierarchical organization, cancer cells can temporarily adapt a quiescent and drug-resistant state, which, similar to adult stem cells, may be regulated by microenvironmental cues, including therapeutic regimens [
      • Batlle E
      • Clevers H
      Cancer stem cells revisited.
      ,
      • Essers MA
      • Trumpp A
      Targeting leukemic stem cells by breaking their dormancy.
      ]. Evidence that ALL blasts survive chemotherapy by adapting a quiescent state in response to microenvironmental cues has been provided in preclinical models [
      • Boyerinas B
      • Zafrir M
      • Yesilkanal AE
      • Price TT
      • Hyjek EM
      • Sipkins DA
      Adhesion to osteopontin in the bone marrow niche regulates lymphoblastic leukemia cell dormancy.
      ]. The hypothesis that quiescent therapy-persisting ALL cells may be the source for relapse is further supported by the fact that therapy-resistant cells isolated from patients at MRD are predominantly quiescent [
      • Lutz C
      • Woll PS
      • Hall G
      • et al.
      Quiescent leukaemic cells account for minimal residual disease in childhood lymphoblastic leukaemia.
      ]. Given the accumulating evidence that quiescence may be an attribute of relapse-inducing cells, we set out to develop a model that allowed us to functionally characterize quiescent cells in primary ALL patient samples [
      • Ebinger S
      • Ozdemir EZ
      • Ziegenhain C
      • et al.
      Characterization of rare, dormant, and therapy–resistant cells in acute lymphoblastic leukemia.
      ]. We hypothesized that such a model would allow us to study biological characteristics of relapse-inducing cells in ALL and help to resolve questions of translational importance: Do these traits already exist at diagnosis or are they acquired during therapy? Are these traits reversible or permanent?

      Patient-derived xenograft model to track quiescent ALL cells over the course of therapy

      To be able to track and isolate small numbers of viable patient-derived xenograft (PDX) ALL cells from the bone marrow of mice, we utilized our previously established lentiviral transduction protocols and ectopically expressed luciferase [
      • Terziyska N
      • Castro Alves C
      • Groiss V
      • et al.
      In vivo imaging enables high resolution preclinical trials on patients' leukemia cells growing in mice.
      ], a truncated nerve growth factor receptor and a red fluorophore [
      • Vick B
      • Rothenberg M
      • Sandhofer N
      • et al.
      An advanced preclinical mouse model for acute myeloid leukemia using patients' cells of various genetic subgroups and in vivo bioluminescence imaging.
      ] in PDX ALL blasts. This triple labeling of ALL PDX cells allows us to track their in vivo growth in mice and enables the isolation of minute numbers of viable human ALL cells from the murine bone marrow [
      • Ebinger S
      • Ozdemir EZ
      • Ziegenhain C
      • et al.
      Characterization of rare, dormant, and therapy–resistant cells in acute lymphoblastic leukemia.
      ]. Following one passage in mice for enrichment of transgene-expressing cells, lentivirally transduced PDX ALL cells were stained with carboxyfluorescein succinimidyl ester (CFSE). This fluorescent dye has been useful to track cell divisions of HSC in mice over several months [
      • Takizawa H
      • Regoes RR
      • Boddupalli CS
      • Bonhoeffer S
      • Manz MG
      Dynamic variation in cycling of hematopoietic stem cells in steady state and inflammation.
      ]. CSFE labels cytoplasmic proteins and is thus independent of the cell cycle status, is equally distributed to daughter cells with each cell division, and, importantly, unlike other proliferation dyes (e.g. BrdU), does not require permeabilization of cells for staining [
      • Takizawa H
      • Regoes RR
      • Boddupalli CS
      • Bonhoeffer S
      • Manz MG
      Dynamic variation in cycling of hematopoietic stem cells in steady state and inflammation.
      ]. Therefore, the combined use of lentiviral transduction and CSFE labelling allows for tracking and isolation of nondividing viable cells from the in vivo environment.
      Upon transplantation into immunodeficient recipient mice, we identified ALL cells that retained the CSFE dye up to 3 weeks, shortly before mice had to be sacrificed due to high leukemia burden. We could identify a rare subpopulation of these label-retaining cells (LRCs) that had undergone no more than three cell divisions among all nine transgenic ALL-PDX samples analyzed [
      • Ebinger S
      • Ozdemir EZ
      • Ziegenhain C
      • et al.
      Characterization of rare, dormant, and therapy–resistant cells in acute lymphoblastic leukemia.
      ]. Immunohistochemical analysis revealed that LRCs, unlike non-LRCs, are preferentially found close to the endosteum, a niche that has previously been suggested to regulate quiescence and resistance to therapy of LIC in vivo [
      • Ishikawa F
      • Yoshida S
      • Saito Y
      • et al.
      Chemotherapy-resistant human AML stem cells home to and engraft within the bone-marrow endosteal region.
      ,
      • Saito Y
      • Uchida N
      • Tanaka S
      • et al.
      Induction of cell cycle entry eliminates human leukemia stem cells in a mouse model of AML.
      ,
      • Boyerinas B
      • Zafrir M
      • Yesilkanal AE
      • Price TT
      • Hyjek EM
      • Sipkins DA
      Adhesion to osteopontin in the bone marrow niche regulates lymphoblastic leukemia cell dormancy.
      ,
      • Pal D
      • Heidenreich O
      • Vormoor J
      Dormancy stems the tide of chemotherapy.
      ].

      Functional characterization of LRC in ALL-PDX

      Given the putative link among quiescence, stemness, and chemoresistance, we first characterized the leukemia-initiating potential of LRCs and evaluated how they respond to chemotherapy [
      • Ebinger S
      • Ozdemir EZ
      • Ziegenhain C
      • et al.
      Characterization of rare, dormant, and therapy–resistant cells in acute lymphoblastic leukemia.
      ]. LIC frequency was very similar between the LRC and non-LRC compartments, which is in agreement with the stochastic stem cell model proposed for ALL, wherein most cells are capable of propagating the tumor [
      • Elder A
      • Bomken S
      • Wilson I
      • et al.
      Abundant and equipotent founder cells establish and maintain acute lymphoblastic leukaemia.
      ]. However, these results demonstrate phenotypic heterogeneity within the LIC compartment, wherein a rare subpopulation of LICs is quiescent. To test whether treatment resistance and dormancy are linked, we treated leukemia-bearing mice with chemotherapy, which strongly selected for LRC, whereas most non-LRCs were eradicated [
      • Ebinger S
      • Ozdemir EZ
      • Ziegenhain C
      • et al.
      Characterization of rare, dormant, and therapy–resistant cells in acute lymphoblastic leukemia.
      ]. Importantly, surviving LRCs retained the ability to propagate the leukemia upon retransplantation into secondary mice and formed tumors with similar growth kinetics as untreated LRC. Therefore, LRCs share the combined features of dormancy, drug resistance, and stemness, indicating that these cells may serve as preclinical surrogates for relapse-inducing cells in ALL [
      • Ebinger S
      • Ozdemir EZ
      • Ziegenhain C
      • et al.
      Characterization of rare, dormant, and therapy–resistant cells in acute lymphoblastic leukemia.
      ] (Figure 1).
      Figure 1.
      Figure 1Plasticity of ALL-LICs. Characterization of LRCs in ALL PDXs revealed that LICs harbor functional plasticity and exist in at least two states within the bone marrow microenvironment: a proliferative, chemosensitive state and a dormant, chemoresistant state. The adverse phenotypes of dormancy and therapy resistance are reversible upon release from the bone marrow microenvironment, whereas stemness remains a constant feature. Single-cell RNA sequencing analysis demonstrated that the rare subpopulation of LRCs is quiescent and the most upregulated pathway is cell adhesion, indicating that the interaction with the microenvironment might influence the dormant phenotype.
      To further characterize molecular features of LRCs and to strengthen the link to MRD, LRCs and MRD cells established from our PDX model (PDX-MRD cells) were subjected to single-cell RNA sequencing. Gene expression profiles of single or bulk leukemia cells of untreated LRCs and PDX-MRD cells demonstrated that overall RNA content was decreased in LRCs, which is indicative of a low metabolic activity, a defining characteristic of dormant cells. Furthermore, in both LRCs and PDX-MRD cells, cell cycle and DNA replication were the most downregulated pathways. Therefore, transcriptomic analysis reinforced the presence of a dormancy phenotype in LRCs and PDX-MRD cells (Figure 1). Interestingly, the most upregulated gene network in LRCs and PDX-MRD cells was cell adhesion, indicating that the interaction with the microenvironment might mediate the observed phenotypes [
      • Ebinger S
      • Ozdemir EZ
      • Ziegenhain C
      • et al.
      Characterization of rare, dormant, and therapy–resistant cells in acute lymphoblastic leukemia.
      ] (Figure 1). The established LRC signature was also identified in primary MRD cells isolated from adult or pediatric patients and differentially regulated genes of published transcriptomes of CD34-positive chronic myeloid leukemia cells, acute leukemia stem cells, hematopoietic stem cells, and pediatric ALL cells with high risk of relapse were significantly enriched in LRCs [
      • Ebinger S
      • Ozdemir EZ
      • Ziegenhain C
      • et al.
      Characterization of rare, dormant, and therapy–resistant cells in acute lymphoblastic leukemia.
      ]. Importantly, the phenotypes of both dormancy and therapy resistance of LRCs were reversible: when LRCs and non-LRCs were transplanted into secondary mice, LRCs gave rise to tumors with similar growth kinetics as non-LRCs and vice versa; non-LRCs gave rise to tumors that contained a quiescent subpopulation at a similar frequency as observed following transplantation of unsorted leukemic cells (Figure 1). When LRCs or non-LRCs were treated with chemotherapeutic drugs in vitro, they demonstrated similar cell death rates, indicating that the release from the bone marrow microenvironment sensitizes LRCs to therapy. Accordingly, coculture of LRCs, non-LRCs, or unsorted bulk cells with feeder cells reduced drug sensitivity, strongly suggesting a role for the bone marrow microenvironment in determining drug resistance.
      In summary, our studies demonstrated that there is significant functional heterogeneity within the abundant LIC population in ALL, wherein only a rare subpopulation of ALL-LICs displays the adverse phenotypes of quiescence and drug resistance. These LRCs exist before the onset of therapy and their adverse phenotypes are reversible. Additionally, the high similarity of LRCs and MRD cells isolated from pediatric and adult patients strongly suggests that LRCs can be used as surrogates for relapse-inducing cells in patients [
      • Ebinger S
      • Ozdemir EZ
      • Ziegenhain C
      • et al.
      Characterization of rare, dormant, and therapy–resistant cells in acute lymphoblastic leukemia.
      ,
      • Pal D
      • Heidenreich O
      • Vormoor J
      Dormancy stems the tide of chemotherapy.
      ]. The presence of a quiescent and therapy-resistant subpopulation of LICs in ALL has important implications for therapy. In particular, the fact the adverse phenotypes of LICs are reversible once released from the protective bone marrow environment offers therapeutic opportunities.

      Therapeutic implications

      Environment-mediated drug resistance provides an explanation for the clinical observation that maintenance therapy with low-dose oral chemotherapy for about 2 years is required, even in low-risk patient groups, to prevent relapse in ALL [
      • Schrappe M
      • Reiter A
      • Zimmermann M
      • et al.
      Long-term results of four consecutive trials in childhood ALL performed by the ALL-BFM study group from 1981 to 1995. Berlin-Frankfurt-Munster.
      ]. However, maintenance therapy is not always sufficient and relapse might occur under treatment. Because even patients who maintained complete remission over several years can relapse, targeting long-term dormant residual cells represents an important unmet clinical need to increase the chance for long-term remission and cure (Figure 2).
      Figure 2.
      Figure 2Rationale and possible approaches for targeting dormant, therapy-resistant ALL-LICs. The characterization of ALL-PDX models during the course of therapy indicated that quiescent LRCs preferably survive chemotherapy and persist at MRD. Dormant cells are already present at the time of diagnosis. Occasional transitions from the quiescent into a chemosensitive, proliferative state (dashed arrow) may explain that long-term maintenance therapy is required to prevent relapse in ALL patients. However, reversibility of the quiescent phenotype (red arrows) allows these LICs to resume proliferation at later time points and proliferate into relapse. Accordingly, therapeutic targeting of this dormant subpopulation of LICs may be necessary and sufficient to prevent the development of relapse in ALL. Possible routes to target therapy-persisting LICs are depicted in the lower left box. Inhibition of LIC stroma, a LIC–extracellular matrix (ECM) interaction, downstream adhesion signaling, or interfering with soluble factors that home LICs into the bone marrow microenvironment may release cells from the protective niche and render them sensitive to standard chemotherapy. Metabolic vulnerabilities of quiescent LICs provide an alternative target, which may allow eradication of LICs without the need to induce their proliferation. To prevent the de novo generation of quiescent, drug-resistant LICs during the course of therapy, epigenetic programs could be targeted. Some putative drugs to achieve each goal are depicted.
      Multiple routes can be envisioned to target LRCs (Figure 2). It has long been known that leukemic cells depend on stromal support for growth and survival, which is mediated by direct adhesion and soluble factors that can affect treatment response [
      • Polak R
      • de Rooij B
      • Pieters R
      • den Boer ML
      B-cell precursor acute lymphoblastic leukemia cells use tunneling nanotubes to orchestrate their microenvironment.
      ,
      • Iwamoto S
      • Mihara K
      • Downing JR
      • Pui CH
      • Campana D
      Mesenchymal cells regulate the response of acute lymphoblastic leukemia cells to asparaginase.
      ,
      • Duan CW
      • Shi J
      • Chen J
      • et al.
      Leukemia-propagating cells rebuild an evolving niche in response to therapy.
      ,
      • Boyerinas B
      • Zafrir M
      • Yesilkanal AE
      • Price TT
      • Hyjek EM
      • Sipkins DA
      Adhesion to osteopontin in the bone marrow niche regulates lymphoblastic leukemia cell dormancy.
      ,
      • Cahu X
      • Calvo J
      • Poglio S
      • et al.
      Bone marrow sites differently imprint dormancy and chemoresistance to T-cell acute lymphoblastic leukemia.
      ,
      • Manabe A
      • Coustan-Smith E
      • Behm FG
      • Raimondi SC
      • Campana D
      Bone marrow-derived stromal cells prevent apoptotic cell death in B-lineage acute lymphoblastic leukemia.
      ,
      • Boutter J
      • Huang Y
      • Marovca B
      • et al.
      Image-based RNA interference screening reveals an individual dependence of acute lymphoblastic leukemia on stromal cysteine support.
      ,
      • Juarez J
      • Dela Pena A
      • Baraz R
      • et al.
      CXCR4 antagonists mobilize childhood acute lymphoblastic leukemia cells into the peripheral blood and inhibit engraftment.
      ,
      • Pitt LA
      • Tikhonova AN
      • Hu H
      • et al.
      CXCL12-producing vascular endothelial niches control acute T cell leukemia maintenance.
      ,
      • Manabe A
      • Murti KG
      • Coustan-Smith E
      • et al.
      Adhesion-dependent survival of normal and leukemic human B lymphoblasts on bone marrow stromal cells.
      ] and our data also strongly suggest that the release of ALL cells from the bone marrow environment sensitizes leukemic blasts to conventional therapies [
      • Ebinger S
      • Ozdemir EZ
      • Ziegenhain C
      • et al.
      Characterization of rare, dormant, and therapy–resistant cells in acute lymphoblastic leukemia.
      ]. Inspired by the discovery of agents that can activate dormant HSCs [
      • Essers MA
      • Trumpp A
      Targeting leukemic stem cells by breaking their dormancy.
      ], blocking the CXCL12/CXCR4 axis, inhibition of osteopontin, cell adhesion molecules, and treatment with granulocyte colony-stimulating factor (G-CSF) have been demonstrated to affect the homing of leukemic cells to the bone marrow and influence therapeutic response in experimental models [
      • Saito Y
      • Uchida N
      • Tanaka S
      • et al.
      Induction of cell cycle entry eliminates human leukemia stem cells in a mouse model of AML.
      ,
      • Boyerinas B
      • Zafrir M
      • Yesilkanal AE
      • Price TT
      • Hyjek EM
      • Sipkins DA
      Adhesion to osteopontin in the bone marrow niche regulates lymphoblastic leukemia cell dormancy.
      ,
      • Pitt LA
      • Tikhonova AN
      • Hu H
      • et al.
      CXCL12-producing vascular endothelial niches control acute T cell leukemia maintenance.
      ,
      • Passaro D
      • Irigoyen M
      • Catherinet C
      • et al.
      CXCR4 is required for leukemia-initiating cell activity in T cell acute lymphoblastic leukemia.
      ,
      • Sipkins DA
      • Wei X
      • Wu JW
      • et al.
      In vivo imaging of specialized bone marrow endothelial microdomains for tumour engraftment.
      ,
      • Jacamo R
      • Chen Y
      • Wang Z
      • et al.
      Reciprocal leukemia-stroma VCAM-1/VLA-4-dependent activation of NF-kappaB mediates chemoresistance.
      ] (Figure 2). Plerixafor and G-CSF, which are approved for HSC mobilization, or natalizumab, a monoclonal antibody that blocks integrin α4, are promising candidates to evaluate for LRC-specific combination therapy [
      • Cooper TM
      • Sison EAR
      • Baker SD
      • et al.
      A phase 1 study of the CXCR4 antagonist plerixafor in combination with high-dose cytarabine and etoposide in children with relapsed or refractory acute leukemias or myelodysplastic syndrome. A Pediatric Oncology Experimental Therapeutics Investigators' Consortium study (POE 10–03).
      ,
      • Giebel S
      • Thomas X
      • Hallbook H
      • et al.
      The prophylactic use of granulocyte-colony stimulating factor during remission induction is associated with increased leukaemia-free survival of adults with acute lymphoblastic leukaemia: A joint analysis of five randomised trials on behalf of the EWALL.
      ,
      • Uy GL
      • Hsu YM
      • Schmidt AP
      • et al.
      Targeting bone marrow lymphoid niches in acute lymphoblastic leukemia.
      ,
      • Welschinger R
      • Liedtke F
      • Basnett J
      • et al.
      Plerixafor (AMD3100) induces prolonged mobilization of acute lymphoblastic leukemia cells and increases the proportion of cycling cells in the blood in mice.
      ,
      • Hsieh YT
      • Gang EJ
      • Geng H
      • et al.
      Integrin alpha4 blockade sensitizes drug resistant pre-B acute lymphoblastic leukemia to chemotherapy.
      ]. However, because leukemic growth and chemotherapy induces massive remodeling of the stromal bone marrow environment [
      • Duan CW
      • Shi J
      • Chen J
      • et al.
      Leukemia-propagating cells rebuild an evolving niche in response to therapy.
      ,
      • Colmone A
      • Amorim M
      • Pontier AL
      • Wang S
      • Jablonski E
      • Sipkins DA
      Leukemic cells create bone marrow niches that disrupt the behavior of normal hematopoietic progenitor cells.
      ,
      • Schepers K
      • Pietras EM
      • Reynaud D
      • et al.
      Myeloproliferative neoplasia remodels the endosteal bone marrow niche into a self–reinforcing leukemic niche.
      ,
      • Medyouf H
      • Mossner M
      • Jann JC
      • et al.
      Myelodysplastic cells in patients reprogram mesenchymal stromal cells to establish a transplantable stem cell niche disease unit.
      ,
      • Duarte D
      • Hawkins ED
      • Akinduro O
      • et al.
      Inhibition of endosteal vascular niche remodeling rescues hematopoietic stem cell loss in AML.
      ], signals that regulate LIC homing likely differ from those regulating HSCs. The remodeled niches can furthermore be specific for distinct leukemia subtypes [
      • Duarte D
      • Hawkins ED
      • Akinduro O
      • et al.
      Inhibition of endosteal vascular niche remodeling rescues hematopoietic stem cell loss in AML.
      ,
      • Hawkins ED
      • Duarte D
      • Akinduro O
      • et al.
      T-cell acute leukaemia exhibits dynamic interactions with bone marrow microenvironments.
      ] and may change over the course of disease progression or following therapy. Accordingly, leukemia-specific signals will need to be identified to efficiently induce cell cycle entry of all leukemic blasts. In this context, CRISPR-Cas9-edited NGS mice may serve useful in elucidating the role of individual niche-derived factors [
      • Tirado-Gonzalez I
      • Czlonka E
      • Nevmerzhitskaya A
      • et al.
      CRISPR/Cas9-edited NSG mice as PDX models of human leukemia to address the role of niche-derived SPARC.
      ]. Another point to consider is that real-time in vivo monitoring of T-ALL cells within the bone marrow demonstrated a highly motile phenotype of ALL blasts, suggesting that leukemic cells may not permanently engraft in a specific niche [
      • Hawkins ED
      • Duarte D
      • Akinduro O
      • et al.
      T-cell acute leukaemia exhibits dynamic interactions with bone marrow microenvironments.
      ]. Therefore, interactions between leukemic blasts and their environment are versatile and multiple leukemia-specific factors may need to be targeted therapeutically to achieve an efficient release of all blasts from the bone marrow. Alternatively, one may envision targeting leukemia-cell-intrinsic adhesion pathways, with one putative target being focal adhesion kinase (FAK) (Figure 2). Small-molecule FAK inhibitors such as defactinib or GSK2256098 are currently under clinical investigation in solid tumors [
      • Soria JC
      • Gan HK
      • Blagden SP
      • et al.
      A phase I, pharmacokinetic and pharmacodynamic study of GSK2256098, a focal adhesion kinase inhibitor, in patients with advanced solid tumors.
      ,
      • Wang-Gillam A
      • Lockhart AC
      • Tan BR
      • et al.
      Phase I study of defactinib combined with pembrolizumab and gemcitabine in patients with advanced cancer.
      ] and can be tested for their ability to sensitize LRCs to chemotherapy. Inhibition of FAK signaling has recently been demonstrated to disrupt cell adhesion in highly aggressive Philadelphia-chromosome-positive, IKZF1-mutated ALL mouse models and FAK inhibition combined with a BCR-ABL1 inhibitor effectively abrogated leukemic cell growth in vivo [
      • Churchman ML
      • Evans K
      • Richmond J
      • et al.
      Synergism of FAK and tyrosine kinase inhibition in Ph(+) B–ALL.
      ].
      Instead of addressing the interaction between leukemic blasts and the environment, direct targeting of quiescent cells is another possibility to eradicate long-term persisting LICs (Figure 2). Metabolic vulnerabilities of quiescent drug-tolerant persisting cells have been identified and targeting, for example, their reliance on either oxidative phosphorylation (OXPHOS) [
      • Roesch A
      • Fukunaga-Kalabis M
      • Schmidt EC
      • et al.
      A temporarily distinct subpopulation of slow–cycling melanoma cells is required for continuous tumor growth.
      ,
      • Roesch A
      • Vultur A
      • Bogeski I
      • et al.
      Overcoming intrinsic multidrug resistance in melanoma by blocking the mitochondrial respiratory chain of slow-cycling JARID1B(high) cells.
      ,
      • Viale A
      • Pettazzoni P
      • Lyssiotis CA
      • et al.
      Oncogene ablation-resistant pancreatic cancer cells depend on mitochondrial function.
      ] or an increased antioxidant capacity [
      • Oshimori N
      • Oristian D
      • Fuchs E
      TGF-beta promotes heterogeneity and drug resistance in squamous cell carcinoma.
      ] may eradicate these cells or render them sensitive to conventional therapy. The bone marrow microenvironment also mediates metabolic adaptions of ALL blasts that support their survival [
      • Boutter J
      • Huang Y
      • Marovca B
      • et al.
      Image-based RNA interference screening reveals an individual dependence of acute lymphoblastic leukemia on stromal cysteine support.
      ] and microenvironment-mediated redox adaptions in ALL blasts reportedly mediate therapy resistance in cell culture models [
      • Liu J
      • Masurekar A
      • Johnson S
      • et al.
      Stromal cell-mediated mitochondrial redox adaptation regulates drug resistance in childhood acute lymphoblastic leukemia.
      ]. A multitude of drugs that target either OXPHOS or inhibit antioxidant production are available and have been approved for cancer treatment or non-oncological indications [
      • Ashton TM
      • McKenna WG
      • Kunz-Schughart LA
      • Higgins GS
      Oxidative phosphorylation as an emerging target in cancer therapy.
      ]. Possible drugs to test for their capacity to deplete LRCs can include the OXPHOS inhibitors metformin [
      • Trucco M
      • Barredo JC
      • Goldberg J
      • et al.
      A phase I window, dose escalating and safety trial of metformin in combination with induction chemotherapy in relapsed refractory acute lymphoblastic leukemia: Metformin with induction chemotherapy of vincristine, dexamethasone, PEG–asparaginase, and doxorubicin.
      ,
      • Hirsch HA
      • Iliopoulos D
      • Tsichlis PN
      • Struhl K
      Metformin selectively targets cancer stem cells, and acts together with chemotherapy to block tumor growth and prolong remission.
      ] and arsenic trioxide [
      • Ashton TM
      • McKenna WG
      • Kunz-Schughart LA
      • Higgins GS
      Oxidative phosphorylation as an emerging target in cancer therapy.
      ]. Furthermore, the anti-rheumatic drugs sulfasalazine and auranofin, together with the GSH-depleting drug buthionine sulfoximine, may serve to sensitize therapy-resistant cells to genotoxic agents [
      • Harris IS
      • Treloar AE
      • Inoue S
      • et al.
      Glutathione and thioredoxin antioxidant pathways synergize to drive cancer initiation and progression.
      ]. The antibiotic tigecycline and inhibition of Bcl-2 antiapoptotic proteins have also been shown to excerpt their antitumor activity by impairing mitochondrial function and increasing oxidative stress [
      • Kuntz EM
      • Baquero P
      • Michie AM
      • et al.
      Targeting mitochondrial oxidative phosphorylation eradicates therapy–resistant chronic myeloid leukemia stem cells.
      ,
      • Lagadinou ED
      • Sach A
      • Callahan K
      • et al.
      BCL-2 inhibition targets oxidative phosphorylation and selectively eradicates quiescent human leukemia stem cells.
      ,
      • Perciavalle RM
      • Stewart DP
      • Koss B
      • et al.
      Anti-apoptotic MCL-1 localizes to the mitochondrial matrix and couples mitochondrial fusion to respiration.
      ]. These findings provide a rationale for testing tigecycline or the BH3 mimetics navitoclax, venetoclax, and S63845[
      • Kotschy A
      • Szlavik Z
      • Murray J
      • et al.
      The MCL1 inhibitor S63845 is tolerable and effective in diverse cancer models.
      ,
      • Boidol B
      • Kornauth C
      • van der Kouwe E
      • et al.
      First-in-human response of BCL-2 inhibitor venetoclax in T-cell prolymphocytic leukemia.
      ,
      • DiNardo CD
      • Pratz KW
      • Letai A
      • et al.
      Safety and preliminary efficacy of venetoclax with decitabine or azacitidine in elderly patients with previously untreated acute myeloid leukaemia: A non-randomised, open-label, phase 1b study.
      ] for their use as LRC-specific therapies.
      Finally, interfering with the epigenetic programs that allow persisting cells to reversibly enter/escape a quiescent and therapy resistant state may be of therapeutic merit (Figure 2), as demonstrated in diverse cancer cell lines and a T-ALL xenograft model [
      • Knoechel B
      • Roderick JE
      • Williamson KE
      • et al.
      An epigenetic mechanism of resistance to targeted therapy in T cell acute lymphoblastic leukemia.
      ,
      • Sharma SV
      • Lee DY
      • Li B
      • et al.
      A chromatin-mediated reversible drug-tolerant state in cancer cell subpopulations.
      ]. Several epigenetic therapeutics are either approved or under clinical evaluation, including BET bromodomain inhibitors [
      • Stathis A
      • Zucca E
      • Bekradda M
      • et al.
      Clinical response of carcinomas harboring the BRD4-NUT oncoprotein to the targeted bromodomain inhibitor OTX015/MK-8628.
      ,
      • Greenblatt SM
      • Nimer SD
      Chromatin modifiers and the promise of epigenetic therapy in acute leukemia.
      ,
      • Berthon C
      • Raffoux E
      • Thomas X
      • et al.
      Bromodomain inhibitor OTX015 in patients with acute leukaemia: A dose-escalation, phase 1 study.
      ].
      One important consideration regarding the plasticity of the therapy-resistant state is the onset and timing of therapeutic approaches. Although our study has clearly demonstrated that a quiescent and therapy-resistant subpopulation of ALL cells exists before the onset of therapy, our experimental setup did not allow us to monitor the de novo emergence of quiescent cells upon treatment initiation [
      • Ebinger S
      • Ozdemir EZ
      • Ziegenhain C
      • et al.
      Characterization of rare, dormant, and therapy–resistant cells in acute lymphoblastic leukemia.
      ]. However, because LRCs can be derived from non-LRCs upon retransplantation into secondary recipients, it is likely that the quiescent and therapy-resistant population can be continuously replenished. If innovative therapeutic strategies succeed in retrieving resistant leukemia cells from protective niches, then sensitive leukemia cells might take over empty slots and acquire a resistant phenotype. Therefore, therapeutic regimens will likely be most effective when administered throughout the course of therapy, as was also suggested by in vitro experiments in non-small-cell lung cancer cell lines, where only continuous administration of HDAC inhibitors prevented the onset of resistance to EGFR inhibitors [
      • Sharma SV
      • Lee DY
      • Li B
      • et al.
      A chromatin-mediated reversible drug-tolerant state in cancer cell subpopulations.
      ].

      Concluding remarks

      After initially successful chemotherapy, relapse frequently jeopardizes the outcome of cancer patients. To improve the prognosis of ALL patients, treatment strategies that eliminate tumor cells at MRD and prevent relapse are urgently required. Although ALL cells lack a clear hierarchical organization, several adverse phenotypes, including quiescence and chemoresistance, are maintained in a rare subset of ALL-LICs, which are otherwise generally attributed to stem cells. The fact that dormancy and resistance are reversible upon release from the microenvironment indicates that transcriptional reprogramming of ALL-LICs is an early event in the generation of therapy resistance. This process may precede the onset of hardwired irreversible resistance and relapse and a similar multistep mechanism of the emergence of resistant disease has recently been suggested under treatment with the immunotoxin moxetumomab pseudotox [
      • Muller F
      • Cunningham T
      • Stookey S
      • et al.
      5-Azacytidine prevents relapse and produces long-term complete remissions in leukemia xenografts treated with moxetumomab pasudotox.
      ]. The in vivo model described in our recent study [
      • Ebinger S
      • Ozdemir EZ
      • Ziegenhain C
      • et al.
      Characterization of rare, dormant, and therapy–resistant cells in acute lymphoblastic leukemia.
      ], in combination with CRISPR-Cas9-edited NGS mice [
      • Tirado-Gonzalez I
      • Czlonka E
      • Nevmerzhitskaya A
      • et al.
      CRISPR/Cas9-edited NSG mice as PDX models of human leukemia to address the role of niche-derived SPARC.
      ], can facilitate study of the regulatory factors within the bone marrow microenvironment that endow LICs with adverse characteristics. Our models can thus serve as preclinical tools with which to test therapeutic strategies that aim to interfere with the interaction between leukemic blasts and their niche to prevent relapse and ultimately improve the prognosis and cure rate of patients with ALL.

      Conflict of interest disclosure

      The authors declare no competing financial interests.

      Acknowledgments

      We apologize to our colleagues whose excellent studies have advanced the field but could not be included in this article due to space limitations.
      This work was supported by grants from the European Research Commission (Consolidator Grant 681524 to IJ); a Mildred Scheel Professorship by German Cancer Aid; and the German Research Foundation (DFG) Collaborative Research Center 1243 “Genetic and Epigenetic Evolution of Hematopoietic Neoplasms” (project A05; DFG proposal MA 1876/13-1; by Bettina Bräu Stiftung and Dr. Helmut Legerlotz Stiftung).

      References

        • Inaba H
        • Greaves M
        • Mullighan CG
        Acute lymphoblastic leukaemia.
        Lancet. 2013; 381: 1943-1955
        • Dohner H
        • Weisdorf DJ
        • Bloomfield CD
        Acute myeloid leukemia.
        N Engl J Med. 2015; 373: 1136-1152
        • Luskin MR
        • Murakami MA
        • Manalis SR
        • Weinstock DM
        Targeting minimal residual disease. a path to cure?.
        Nat Rev Cancer. 2018; 18: 255-263
        • Kreso A
        • Dick JE
        Evolution of the cancer stem cell model.
        Cell Stem Cell. 2014; 14: 275-291
        • Batlle E
        • Clevers H
        Cancer stem cells revisited.
        Nat Med. 2017; 23: 1124-1134
        • Laurenti E
        • Gottgens B
        From haematopoietic stem cells to complex differentiation landscapes.
        Nature. 2018; 553: 418-426
        • Crane GM
        • Jeffery E
        • Morrison SJ
        Adult haematopoietic stem cell niches.
        Nat Rev Immunol. 2017; 17: 573-590
        • Trumpp A
        • Essers M
        • Wilson A
        Awakening dormant haematopoietic stem cells.
        Nat Rev Immunol. 2010; 10: 201-209
        • Wilson A
        • Laurenti E
        • Oser G
        • et al.
        Hematopoietic stem cells reversibly switch from dormancy to self–renewal during homeostasis and repair.
        Cell. 2008; 135: 1118-1129
        • Foudi A
        • Hochedlinger K
        • Van Buren D
        • et al.
        Analysis of histone 2B-GFP retention reveals slowly cycling hematopoietic stem cells.
        Nat Biotechnol. 2009; 27: 84-90
        • Qiu J
        • Papatsenko D
        • Niu X
        • Schaniel C
        • Moore K
        Divisional history and hematopoietic stem cell function during homeostasis.
        Stem Cell Rep. 2014; 2: 473-490
        • Bernitz JM
        • Kim HS
        • MacArthur B
        • Sieburg H
        • Moore K
        Hematopoietic stem cells count and remember self-renewal divisions.
        Cell. 2016; 167 (e1210): 1296-1309
        • Cabezas-Wallscheid N
        • Buettner F
        • Sommerkamp P
        • et al.
        Vitamin A-retinoic acid signaling regulates hematopoietic stem cell dormancy.
        Cell. 2017; 169 (e819): 807-823
        • Takizawa H
        • Regoes RR
        • Boddupalli CS
        • Bonhoeffer S
        • Manz MG
        Dynamic variation in cycling of hematopoietic stem cells in steady state and inflammation.
        J Exp Med. 2011; 208: 273-284
        • Boettcher S
        • Manz MG
        Regulation of inflammation- and infection-driven hematopoiesis.
        Trends Immunol. 2017; 38: 345-357
        • Randall TD
        • Weissman IL
        Phenotypic and functional changes induced at the clonal level in hematopoietic stem cells after 5-fluorouracil treatment.
        Blood. 1997; 89: 3596-3606
        • Lapidot T
        • Sirard C
        • Vormoor J
        • et al.
        A cell initiating human acute myeloid leukaemia after transplantation into SCID mice.
        Nature. 1994; 367: 645-648
        • Bonnet D
        • Dick JE
        Human acute myeloid leukemia is organized as a hierarchy that originates from a primitive hematopoietic cell.
        Nat Med. 1997; 3: 730-737
        • Dick JE
        Stem cell concepts renew cancer research.
        Blood. 2008; 112: 4793-4807
        • Hope KJ
        • Jin L
        • Dick JE
        Acute myeloid leukemia originates from a hierarchy of leukemic stem cell classes that differ in self-renewal capacity.
        Nat Immunol. 2004; 5: 738-743
        • Guan Y
        • Gerhard B
        • Hogge DE
        Detection, isolation, and stimulation of quiescent primitive leukemic progenitor cells from patients with acute myeloid leukemia (AML).
        Blood. 2003; 101: 3142-3149
        • Ng SW
        • Mitchell A
        • Kennedy JA
        • et al.
        A 17-gene stemness score for rapid determination of risk in acute leukaemia.
        Nature. 2016; 540: 433-437
        • van Rhenen A
        • Feller N
        • Kelder A
        • et al.
        High stem cell frequency in acute myeloid leukemia at diagnosis predicts high minimal residual disease and poor survival.
        Clin Cancer Res. 2005; 11: 6520-6527
        • Ho TC
        • LaMere M
        • Stevens BM
        • et al.
        Evolution of acute myelogenous leukemia stem cell properties after treatment and progression.
        Blood. 2016; 128: 1671-1678
        • Fong CY
        • Gilan O
        • Lam EY
        • et al.
        BET inhibitor resistance emerges from leukaemia stem cells.
        Nature. 2015; 525: 538-542
        • Shlush LI
        • Mitchell A
        • Heisler L
        • et al.
        Tracing the origins of relapse in acute myeloid leukaemia to stem cells.
        Nature. 2017; 547: 104-108
        • Ishikawa F
        • Yoshida S
        • Saito Y
        • et al.
        Chemotherapy-resistant human AML stem cells home to and engraft within the bone-marrow endosteal region.
        Nat Biotechnol. 2007; 25: 1315-1321
        • Saito Y
        • Uchida N
        • Tanaka S
        • et al.
        Induction of cell cycle entry eliminates human leukemia stem cells in a mouse model of AML.
        Nat Biotechnol. 2010; 28: 275-280
        • Essers MA
        • Trumpp A
        Targeting leukemic stem cells by breaking their dormancy.
        Mol Oncol. 2010; 4: 443-450
        • Kong Y
        • Yoshida S
        • Saito Y
        • et al.
        CD34+CD38+CD19+ as well as CD34+CD38–CD19+ cells are leukemia-initiating cells with self-renewal capacity in human B-precursor ALL.
        Leukemia. 2008; 22: 1207-1213
        • le Viseur C
        • Hotfilder M
        • Bomken S
        • et al.
        In childhood acute lymphoblastic leukemia, blasts at different stages of immunophenotypic maturation have stem cell properties.
        Cancer Cell. 2008; 14: 47-58
        • Rehe K
        • Wilson K
        • Bomken S
        • et al.
        Acute B lymphoblastic leukaemia-propagating cells are present at high frequency in diverse lymphoblast populations.
        EMBO Mol Med. 2013; 5: 38-51
        • Elder A
        • Bomken S
        • Wilson I
        • et al.
        Abundant and equipotent founder cells establish and maintain acute lymphoblastic leukaemia.
        Leukemia. 2017; 31: 2577-2586
        • Anderson K
        • Lutz C
        • van Delft FW
        • et al.
        Genetic variegation of clonal architecture and propagating cells in leukaemia.
        Nature. 2011; 469: 356-361
        • Bardini M
        • Woll PS
        • Corral L
        • et al.
        Clonal variegation and dynamic competition of leukemia–initiating cells in infant acute lymphoblastic leukemia with MLL rearrangement.
        Leukemia. 2015; 29: 38-50
        • Notta F
        • Mullighan CG
        • Wang JC
        • et al.
        Evolution of human BCR-ABL1 lymphoblastic leukaemia–initiating cells.
        Nature. 2011; 469: 362-367
        • Ding LW
        • Sun QY
        • Tan KT
        • et al.
        Mutational landscape of pediatric acute lymphoblastic leukemia.
        Cancer Res. 2017; 77: 390-400
        • Ma X
        • Edmonson M
        • Yergeau D
        • et al.
        Rise and fall of subclones from diagnosis to relapse in pediatric B–acute lymphoblastic leukaemia.
        Nat Commun. 2015; 6: 6604
        • Mullighan CG
        • Phillips LA
        • Su X
        • et al.
        Genomic analysis of the clonal origins of relapsed acute lymphoblastic leukemia.
        Science. 2008; 322: 1377-1380
        • Oshima K
        • Khiabanian H
        • da Silva-Almeida AC
        • et al.
        Mutational landscape, clonal evolution patterns, and role of RAS mutations in relapsed acute lymphoblastic leukemia.
        Proc Natl Acad Sci U S A. 2016; 113: 11306-11311
        • Meyer JA
        • Wang J
        • Hogan LE
        • et al.
        Relapse-specific mutations in NT5C2 in childhood acute lymphoblastic leukemia.
        Nat Genet. 2013; 45: 290-294
        • Tzoneva G
        • Perez-Garcia A
        • Carpenter Z
        • et al.
        Activating mutations in the NT5C2 nucleotidase gene drive chemotherapy resistance in relapsed ALL.
        Nat Med. 2013; 19: 368-371
        • Li B
        • Li H
        • Bai Y
        • et al.
        Negative feedback-defective PRPS1 mutants drive thiopurine resistance in relapsed childhood ALL.
        Nat Med. 2015; 21: 563-571
        • Mullighan CG
        • Zhang J
        • Kasper LH
        • et al.
        CREBBP mutations in relapsed acute lymphoblastic leukaemia.
        Nature. 2011; 471: 235-239
        • Irving J
        • Matheson E
        • Minto L
        • et al.
        Ras pathway mutations are prevalent in relapsed childhood acute lymphoblastic leukemia and confer sensitivity to MEK inhibition.
        Blood. 2014; 124: 3420-3430
        • Molteni CG
        • Te Kronnie G
        • Bicciato S
        • et al.
        PTPN11 mutations in childhood acute lymphoblastic leukemia occur as a secondary event associated with high hyperdiploidy.
        Leukemia. 2010; 24: 232-235
        • Grobner SN
        • Worst BC
        • Weischenfeldt J
        • et al.
        The landscape of genomic alterations across childhood cancers.
        Nature. 2018; 555: 321-327
        • Ma X
        • Liu Y
        • Liu Y
        • et al.
        Pan-cancer genome and transcriptome analyses of 1,699 paediatric leukaemias and solid tumours.
        Nature. 2018; 555: 371-376
        • Spinella JF
        • Richer C
        • Cassart P
        • Ouimet M
        • Healy J
        • Sinnett D
        Mutational dynamics of early and late relapsed childhood ALL: Rapid clonal expansion and long-term dormancy.
        Blood Adv. 2018; 2: 177-188
        • Kunz JB
        • Rausch T
        • Bandapalli OR
        • et al.
        Pediatric T-cell lymphoblastic leukemia evolves into relapse by clonal selection, acquisition of mutations and promoter hypomethylation.
        Haematologica. 2015; 100: 1442-1450
        • Ford AM
        • Mansur MB
        • Furness CL
        • et al.
        Protracted dormancy of pre-leukemic stem cells.
        Leukemia. 2015; 29: 2202-2207
        • Meacham CE
        • Morrison SJ
        Tumour heterogeneity and cancer cell plasticity.
        Nature. 2013; 501: 328-337
        • Meads MB
        • Gatenby RA
        • Dalton WS
        Environment-mediated drug resistance: A major contributor to minimal residual disease.
        Nat Rev Cancer. 2009; 9: 665-674
        • Knoechel B
        • Roderick JE
        • Williamson KE
        • et al.
        An epigenetic mechanism of resistance to targeted therapy in T cell acute lymphoblastic leukemia.
        Nat Genet. 2014; 46: 364-370
        • Kreso A
        • O'Brien CA
        • van Galen P
        • et al.
        Variable clonal repopulation dynamics influence chemotherapy response in colorectal cancer.
        Science. 2013; 339: 543-548
        • Roesch A
        • Fukunaga-Kalabis M
        • Schmidt EC
        • et al.
        A temporarily distinct subpopulation of slow–cycling melanoma cells is required for continuous tumor growth.
        Cell. 2010; 141: 583-594
        • Roesch A
        • Vultur A
        • Bogeski I
        • et al.
        Overcoming intrinsic multidrug resistance in melanoma by blocking the mitochondrial respiratory chain of slow-cycling JARID1B(high) cells.
        Cancer Cell. 2013; 23: 811-825
        • Sharma SV
        • Lee DY
        • Li B
        • et al.
        A chromatin-mediated reversible drug-tolerant state in cancer cell subpopulations.
        Cell. 2010; 141: 69-80
        • Viale A
        • Pettazzoni P
        • Lyssiotis CA
        • et al.
        Oncogene ablation-resistant pancreatic cancer cells depend on mitochondrial function.
        Nature. 2014; 514: 628-632
        • Polak R
        • de Rooij B
        • Pieters R
        • den Boer ML
        B-cell precursor acute lymphoblastic leukemia cells use tunneling nanotubes to orchestrate their microenvironment.
        Blood. 2015; 126: 2404-2414
        • Iwamoto S
        • Mihara K
        • Downing JR
        • Pui CH
        • Campana D
        Mesenchymal cells regulate the response of acute lymphoblastic leukemia cells to asparaginase.
        J Clin Invest. 2007; 117: 1049-1057
        • Duan CW
        • Shi J
        • Chen J
        • et al.
        Leukemia-propagating cells rebuild an evolving niche in response to therapy.
        Cancer Cell. 2014; 25: 778-793
        • Boyerinas B
        • Zafrir M
        • Yesilkanal AE
        • Price TT
        • Hyjek EM
        • Sipkins DA
        Adhesion to osteopontin in the bone marrow niche regulates lymphoblastic leukemia cell dormancy.
        Blood. 2013; 121: 4821-4831
        • Cahu X
        • Calvo J
        • Poglio S
        • et al.
        Bone marrow sites differently imprint dormancy and chemoresistance to T-cell acute lymphoblastic leukemia.
        Blood Adv. 2017; 1: 1760-1772
        • Chen J
        • Li Y
        • Yu TS
        • et al.
        A restricted cell population propagates glioblastoma growth after chemotherapy.
        Nature. 2012; 488: 522-526
        • Oshimori N
        • Oristian D
        • Fuchs E
        TGF-beta promotes heterogeneity and drug resistance in squamous cell carcinoma.
        Cell. 2015; 160: 963-976
        • Lutz C
        • Woll PS
        • Hall G
        • et al.
        Quiescent leukaemic cells account for minimal residual disease in childhood lymphoblastic leukaemia.
        Leukemia. 2013; 27: 1204-1207
        • Ebinger S
        • Ozdemir EZ
        • Ziegenhain C
        • et al.
        Characterization of rare, dormant, and therapy–resistant cells in acute lymphoblastic leukemia.
        Cancer Cell. 2016; 30: 849-862
        • Terziyska N
        • Castro Alves C
        • Groiss V
        • et al.
        In vivo imaging enables high resolution preclinical trials on patients' leukemia cells growing in mice.
        PLoS One. 2012; 7: e52798
        • Vick B
        • Rothenberg M
        • Sandhofer N
        • et al.
        An advanced preclinical mouse model for acute myeloid leukemia using patients' cells of various genetic subgroups and in vivo bioluminescence imaging.
        PLoS One. 2015; 10e0120925
        • Pal D
        • Heidenreich O
        • Vormoor J
        Dormancy stems the tide of chemotherapy.
        Cancer Cell. 2016; 30: 825-826
        • Schrappe M
        • Reiter A
        • Zimmermann M
        • et al.
        Long-term results of four consecutive trials in childhood ALL performed by the ALL-BFM study group from 1981 to 1995. Berlin-Frankfurt-Munster.
        Leukemia. 2000; 14: 2205-2222
        • Manabe A
        • Coustan-Smith E
        • Behm FG
        • Raimondi SC
        • Campana D
        Bone marrow-derived stromal cells prevent apoptotic cell death in B-lineage acute lymphoblastic leukemia.
        Blood. 1992; 79: 2370-2377
        • Boutter J
        • Huang Y
        • Marovca B
        • et al.
        Image-based RNA interference screening reveals an individual dependence of acute lymphoblastic leukemia on stromal cysteine support.
        Oncotarget. 2014; 5: 11501-11512
        • Juarez J
        • Dela Pena A
        • Baraz R
        • et al.
        CXCR4 antagonists mobilize childhood acute lymphoblastic leukemia cells into the peripheral blood and inhibit engraftment.
        Leukemia. 2007; 21: 1249-1257
        • Pitt LA
        • Tikhonova AN
        • Hu H
        • et al.
        CXCL12-producing vascular endothelial niches control acute T cell leukemia maintenance.
        Cancer Cell. 2015; 27: 755-768
        • Manabe A
        • Murti KG
        • Coustan-Smith E
        • et al.
        Adhesion-dependent survival of normal and leukemic human B lymphoblasts on bone marrow stromal cells.
        Blood. 1994; 83: 758-766
        • Passaro D
        • Irigoyen M
        • Catherinet C
        • et al.
        CXCR4 is required for leukemia-initiating cell activity in T cell acute lymphoblastic leukemia.
        Cancer Cell. 2015; 27: 769-779
        • Sipkins DA
        • Wei X
        • Wu JW
        • et al.
        In vivo imaging of specialized bone marrow endothelial microdomains for tumour engraftment.
        Nature. 2005; 435: 969-973
        • Jacamo R
        • Chen Y
        • Wang Z
        • et al.
        Reciprocal leukemia-stroma VCAM-1/VLA-4-dependent activation of NF-kappaB mediates chemoresistance.
        Blood. 2014; 123: 2691-2702
        • Cooper TM
        • Sison EAR
        • Baker SD
        • et al.
        A phase 1 study of the CXCR4 antagonist plerixafor in combination with high-dose cytarabine and etoposide in children with relapsed or refractory acute leukemias or myelodysplastic syndrome. A Pediatric Oncology Experimental Therapeutics Investigators' Consortium study (POE 10–03).
        Pediatr Blood Cancer. 2017; : 64
        • Giebel S
        • Thomas X
        • Hallbook H
        • et al.
        The prophylactic use of granulocyte-colony stimulating factor during remission induction is associated with increased leukaemia-free survival of adults with acute lymphoblastic leukaemia: A joint analysis of five randomised trials on behalf of the EWALL.
        Eur J Cancer. 2012; 48: 360-367
        • Uy GL
        • Hsu YM
        • Schmidt AP
        • et al.
        Targeting bone marrow lymphoid niches in acute lymphoblastic leukemia.
        Leuk Res. 2015; 39: 1437-1442
        • Welschinger R
        • Liedtke F
        • Basnett J
        • et al.
        Plerixafor (AMD3100) induces prolonged mobilization of acute lymphoblastic leukemia cells and increases the proportion of cycling cells in the blood in mice.
        Exp Hematol. 2013; 41 (.e291): 293-302
        • Hsieh YT
        • Gang EJ
        • Geng H
        • et al.
        Integrin alpha4 blockade sensitizes drug resistant pre-B acute lymphoblastic leukemia to chemotherapy.
        Blood. 2013; 121: 1814-1818
        • Colmone A
        • Amorim M
        • Pontier AL
        • Wang S
        • Jablonski E
        • Sipkins DA
        Leukemic cells create bone marrow niches that disrupt the behavior of normal hematopoietic progenitor cells.
        Science. 2008; 322: 1861-1865
        • Schepers K
        • Pietras EM
        • Reynaud D
        • et al.
        Myeloproliferative neoplasia remodels the endosteal bone marrow niche into a self–reinforcing leukemic niche.
        Cell Stem Cell. 2013; 13: 285-299
        • Medyouf H
        • Mossner M
        • Jann JC
        • et al.
        Myelodysplastic cells in patients reprogram mesenchymal stromal cells to establish a transplantable stem cell niche disease unit.
        Cell Stem Cell. 2014; 14: 824-837
        • Duarte D
        • Hawkins ED
        • Akinduro O
        • et al.
        Inhibition of endosteal vascular niche remodeling rescues hematopoietic stem cell loss in AML.
        Cell Stem Cell. 2018; 22 (e66): 64-77
        • Hawkins ED
        • Duarte D
        • Akinduro O
        • et al.
        T-cell acute leukaemia exhibits dynamic interactions with bone marrow microenvironments.
        Nature. 2016; 538: 518-522
        • Tirado-Gonzalez I
        • Czlonka E
        • Nevmerzhitskaya A
        • et al.
        CRISPR/Cas9-edited NSG mice as PDX models of human leukemia to address the role of niche-derived SPARC.
        Leukemia. 2018; 32: 1049-1052
        • Soria JC
        • Gan HK
        • Blagden SP
        • et al.
        A phase I, pharmacokinetic and pharmacodynamic study of GSK2256098, a focal adhesion kinase inhibitor, in patients with advanced solid tumors.
        Ann Oncol. 2016; 27: 2268-2274
        • Wang-Gillam A
        • Lockhart AC
        • Tan BR
        • et al.
        Phase I study of defactinib combined with pembrolizumab and gemcitabine in patients with advanced cancer.
        J Clin Oncol. 2018; 36 (–380): 380
        • Churchman ML
        • Evans K
        • Richmond J
        • et al.
        Synergism of FAK and tyrosine kinase inhibition in Ph(+) B–ALL.
        JCI Insight. 2016; 1: e86082
        • Liu J
        • Masurekar A
        • Johnson S
        • et al.
        Stromal cell-mediated mitochondrial redox adaptation regulates drug resistance in childhood acute lymphoblastic leukemia.
        Oncotarget. 2015; 6: 43048-43064
        • Ashton TM
        • McKenna WG
        • Kunz-Schughart LA
        • Higgins GS
        Oxidative phosphorylation as an emerging target in cancer therapy.
        Clin Cancer Res. 2018; 24: 2482-2490
        • Trucco M
        • Barredo JC
        • Goldberg J
        • et al.
        A phase I window, dose escalating and safety trial of metformin in combination with induction chemotherapy in relapsed refractory acute lymphoblastic leukemia: Metformin with induction chemotherapy of vincristine, dexamethasone, PEG–asparaginase, and doxorubicin.
        Pediatr Blood Cancer. 2018; 65: e27224
        • Hirsch HA
        • Iliopoulos D
        • Tsichlis PN
        • Struhl K
        Metformin selectively targets cancer stem cells, and acts together with chemotherapy to block tumor growth and prolong remission.
        Cancer Res. 2009; 69: 7507-7511
        • Harris IS
        • Treloar AE
        • Inoue S
        • et al.
        Glutathione and thioredoxin antioxidant pathways synergize to drive cancer initiation and progression.
        Cancer Cell. 2015; 27: 211-222
        • Kuntz EM
        • Baquero P
        • Michie AM
        • et al.
        Targeting mitochondrial oxidative phosphorylation eradicates therapy–resistant chronic myeloid leukemia stem cells.
        Nat Med. 2017; 23: 1234-1240
        • Lagadinou ED
        • Sach A
        • Callahan K
        • et al.
        BCL-2 inhibition targets oxidative phosphorylation and selectively eradicates quiescent human leukemia stem cells.
        Cell Stem Cell. 2013; 12: 329-341
        • Perciavalle RM
        • Stewart DP
        • Koss B
        • et al.
        Anti-apoptotic MCL-1 localizes to the mitochondrial matrix and couples mitochondrial fusion to respiration.
        Nat Cell Biol. 2012; 14: 575-583
        • Kotschy A
        • Szlavik Z
        • Murray J
        • et al.
        The MCL1 inhibitor S63845 is tolerable and effective in diverse cancer models.
        Nature. 2016; 538: 477-482
        • Boidol B
        • Kornauth C
        • van der Kouwe E
        • et al.
        First-in-human response of BCL-2 inhibitor venetoclax in T-cell prolymphocytic leukemia.
        Blood. 2017; 130: 2499-2503
        • DiNardo CD
        • Pratz KW
        • Letai A
        • et al.
        Safety and preliminary efficacy of venetoclax with decitabine or azacitidine in elderly patients with previously untreated acute myeloid leukaemia: A non-randomised, open-label, phase 1b study.
        Lancet Oncol. 2018; 19: 216-228
        • Stathis A
        • Zucca E
        • Bekradda M
        • et al.
        Clinical response of carcinomas harboring the BRD4-NUT oncoprotein to the targeted bromodomain inhibitor OTX015/MK-8628.
        Cancer Discov. 2016; 6: 492-500
        • Greenblatt SM
        • Nimer SD
        Chromatin modifiers and the promise of epigenetic therapy in acute leukemia.
        Leukemia. 2014; 28: 1396-1406
        • Berthon C
        • Raffoux E
        • Thomas X
        • et al.
        Bromodomain inhibitor OTX015 in patients with acute leukaemia: A dose-escalation, phase 1 study.
        Lancet Haematol. 2016; 3: e186-e195
        • Muller F
        • Cunningham T
        • Stookey S
        • et al.
        5-Azacytidine prevents relapse and produces long-term complete remissions in leukemia xenografts treated with moxetumomab pasudotox.
        Proc Natl Acad Sci U S A. 2018; 115: E1867-E1875