CANCER GENOMICS
Tumor Archaeology: Tracking Leukemic Evolution to Its Origins John E. Dick Unearthing of the BRAF mutation in self-renewing hematopoietic stem cells reveals an unexpected origin for hairy cell leukemia—a mature B cell malignancy—and a potential new therapeutic target (Chung et al., this issue).
Cancer arises from the multistep accumulation of mutations, and concerted eforts have yielded an unprecedented understanding of genomic lesions in human cancers (1). Trough the application of single-cell genetic analysis and computational algorithms to uncover genetic diversity from whole-genome sequencing of bulk tumor tissue, it has become clear that cancers are composed of multiple genetically distinct subclones. By applying the tools of population genetics, it has been possible to trace the evolutionary history that created this complex clonal architecture (2). However, these approaches examine only malignant cells, leaving unresolved critical questions regarding the earlier premalignant steps in cancer development. What is the identity of the cell of origin? What is the nature and biological consequences of the initiating genetic lesion? What is the order and number of subsequent mutations needed before cells become overtly malignant? In this issue of Science Translational Medicine, new work by Chung et al. points to hematopoietic stem cells (HSCs) as the potential origin for at least some mature B cell malignancies (3). ANCESTRAL STEPPING STONES For most cancer types, the premalignant phase is clinically silent, making it impossible to address the key questions. However, in acute myeloid leukemia (AML), a subset of cases evolve from a preceding clinically apparent clonal hematological disorder such as a myelodysplastic syndrome or a myeloproliferative neoplasm (4). In many of these cases, the lesion that initiates clonal expansion occurs in HSCs in the bone marrow and is present in all descendent cells, including the AML cells that arise afer acquisition of additional mutations in the founding clone. Princess Margaret Cancer Centre, University Health Network and Department of Molecular Genetics, University of Toronto, Ontario M5G2C1, Canada. E-mail: [email protected]
Tese cases of leukemic progression raise the question of whether some ancestral cell types, bearing only the initiating mutations, persist within the hematopoietic tissues (bone marrow and blood) afer overt leukemia has developed. Te evolutionary history of the leukemia would be hidden in such cells, and analysis of their phenotype and mutations would uncover the “stepping stones” of leukemic progression, which could be followed back to the cell of origin. Te plausibility that leukemic blood samples contain nonleukemic cell types that are ancestral to leukemic blasts was strengthened in recent studies of AML that took advantage of great strides made in our ability to isolate, assay, and characterize pure cell populations that represent the entire human hematopoietic stem and progenitor landscape (5). Collectively, these studies found that peripheral blood and bone marrow samples from patients with AML contain hematopoietic stem and progenitor cells as well as mature T and B cells that bear only a single or a subset of the cancer-associated mutations present in the leukemia blasts (6, 7). Despite the acquisition of mutations, these HSCs retain the ability to diferentiate into a full spectrum of myelo-lymphoid lineages (Fig. 1). However, examination of the proportional contribution that HSCs that carry the initiating mutations made to the overall HSC pool indicates that these mutations confer a competitive growth advantage, leading to clonal expansion. Tese studies established that in a high proportion of AML cases, initiating mutations arise in a primitive cell, likely a HSC, to create a pre-leukemic HSC (preLHSC) that clonally expands. AML evolves from this pool of preL-HSCs through the acquisition of additional genetic alterations. THE ORIGIN OF MALIGNANCIES A key principle to be inferred from the studies described above is that initiating mutations must occur in self-renewing cells
to ensure that a clonally expanded population persists and thus allows for the acquisition of subsequent mutations that lead to malignancy. Mutations in non–self-renewing cells would be purged by diferentiation before additional mutations could be acquired. Once myeloid commitment has occurred, no self-renewing cells remain; thus, in AML, initiating mutations must occur in HSCs. However, in normal lymphoid development, many types of progenitor and mature T and B cells possess a substantial capacity for clonal expansion through antigenic stimulation. Indeed, mature memory B cells retain stem cell–like properties to ensure proper immune function upon future antigen exposure. Tus, many cell types throughout lymphoid maturation could act as putative cells of origin for leukemia or lymphoma development. B cell lymphopoiesis is relatively well understood from a cellular and molecular standpoint; there is a highly ordered gain or loss of specifc cell-surface markers that is coupled with specifc immunoglobulin gene rearrangements and transcription factor expression representing specifc stages of development. Tus, it is tempting to infer the identity of the cell of origin by comparing the blocked development that occurs as a result of malignant transformation to a specifc normal developmental stage. For example, if the cellular, molecular, and signaling phenotype of the malignant cells matches that of a mature plasma cell, this would suggest that the cell of origin is also a mature cell. However, this line of reasoning has several faws. Mutations acquired early might lead to clonal expansion but with retention of normal diferentiation. Only mutations acquired at later stages of leukemogenesis may cause a block in diferentiation that leads to malignancy. In this scenario, the phenotype of the cell that acquires the frst mutation will be completely diferent from the phenotype of the malignant cells. Second, not every leukemia cell is equally able to maintain long-term clonal propagation; some have stem cell–like self-renewal properties. Such leukemia-propagating stem cells have cell-surface markers and growth properties that are distinct from those of bulk malignant cells. If such leukemiapropagating cells happen to be rare, they would be missed when studying bulk cell populations. Te properties of such cells might be more closely related to the cell of origin than to the bulk malignant population.
www.ScienceTranslationalMedicine.org 28 May 2014 Vol 6 Issue 238 238fs23
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FOCUS
Skin Marrow
Hip bone
Normal HSC
B cell expansion Additional mutations
BRAFV600E mutation
HCL-HSC Skewed B cell development
Aberrant self-renewal, clonal expansion
MLP
Granulocytes Dendritic cells/ macrophages CMP Erythrocytes MEP Platelets Reduced myelo-erythroid development GMP
Dendritic T NK cells
MPP
CREDIT: V. ALTOUNIAN/SCIENCE TRANSLATIONAL MEDICINE
Hairy cell
Fig. 1. Backtracking to leukemia’s origin. Acquisition of BRAFV600E mutations within HSCs enhances B cell development and impairs differentiation into other lymphoid and myelo-erythroid blood lineages. The mutated HSCs have increased self-renewal capacity (indicated by thick yellow arrow) resulting in an expanded hairy cell leukemia HSC clone (HCL-HSC) as well as downstream progenitors [multipotential progenitor (MPP) and multilymphoid progenitor (MLP)] that all contain the BRAFV600E mutation. BRAFV600E contributes to enhanced expansion of B cells (indicated by thick black arrows) that then acquire additional mutations, resulting in clinically overt HCL. T, T cell; NK, natural killer cell; CMP, common myeloid progenitor; GMP, granulocyte-macrophage progenitor; MEP, myelo-erythroid precursor.
Indeed, recent evidence suggests that accepted models of development of mature lymphoid malignancies may have been confounded by these issues. For example, recent studies indicate that multiple myeloma, a disease of mature plasma cells, is organized as a hierarchy sustained by rare myelomapropagating cells that are more developmentally immature than the predominant malignant plasma cells; these cells also contribute to proteasome-inhibitor resistance (8).
Genomic analysis of an aggressive subtype of T-ALL (ETP T-ALL) uncovered transcriptional programs and mutations not typically associated with T cell development; rather, mutations were detected in genes that encode proteins that are important for stem cell function, suggesting that cancer-driving mutations originated in a cell type more immature than committed T cells (9). Chronic lymphocytic leukemia (CLL) is a common malignancy characterized by
the clonal expansion of mature CD5+ B cells bearing functional B cell receptors (BCRs) on their surfaces. Antigenic stimulation of these BCRs is typically thought to drive the clonal expansion of a specifc immunoglobulinrearranged cell that might be the cell of origin. However, CLL might be preceded by a B cell lymphocytosis in which multiple subclones are present and only one of these is selected when the disease develops, pointing to an ancestral precursor that had not yet undergone immunoglobulin rearrangement. By sorting bone marrow from CLL patients into various HSC, progenitor, and early B cell lineage populations, provocative evidence has pointed to HSCs and not mature B cells as the origin of the initial cancerdriving mutations (10). Compared with normal HSCs, CLL-HSCs have higher rates of self-renewal and are lymphoid-primed, as evidenced by a higher propensity to generate CD5+ B cells with highly restricted immunoglobulin gene rearrangements typical of the mature CLL cells seen in patients. CLLHSCs also lack translocations found in CLL cells, suggesting that the former cell type is ancestral to the latter. What is missing from these studies is a deep genomic analysis that identifes an initiating mutation that directly implicates HSCs as the cells of origin for malignant CLL B cells. DECONSTRUCTING HAIRY In their study of hairy cell leukemia, Chung et al. now take a substantial leap forward in unveiling HSCs as the cell of origin for at least some mature B cell malignancies (3). Hairy cell leukemia is a chronic lymphoproliferative disorder in which the leukemic hairy cells exhibit classical features of a mature B cell malignancy, including expression of CD19 and surface immunoglobulin and clonal rearrangements of immunoglobulin heavy and light chain genes. In the new work, concerted genome sequencing revealed that virtually all cases of hairy cell leukemia contain a recurrent BRAFV600E mutation. BRAF is a serine/threonine kinase that plays a central role in mitogen-activated protein kinase (MAPK) signaling. Small-molecule inhibitors such as vemurafnib have been developed to block the constitutively active mutant forms of B-RAF. Chung et al. subjected hairy cell leukemia clinical samples to high-resolution fow sorting and isolated populations of phenotypic HSCs, progenitor cells, and mature cells of the hematopoietic lineage. Along with malignant cells, each sorted fraction
www.ScienceTranslationalMedicine.org 28 May 2014 Vol 6 Issue 238 238fs23
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FOCUS
FOCUS
HCL MODELS POINT TO HSC ORIGIN Because it is difcult to carry out mechanistic studies in primary human cells, Chung et al. engineered a number of mouse models in which the BRAFV600E mutation was targeted either to the HSC–progenitor cell compartment or to the B cell lineage alone, with striking results. If BRAFV600E was activated in HSCs or progenitor cells, mice rapidly developed a lethal hematopoietic disorder that was highly similar to HCL and was transplantable into lethally irradiated recipients, which is consistent with a cellautonomous phenotype. In contrast, if the BRAFV600E mutation was targeted to the B cell lineage, no hematopoietic abnormalities were observed for up to 1 year, even though the mutant protein constitutively activated MAPK signaling in B cells. Closer examination of transgenic BRAFV600E mice before the development of overt leukemia demonstrated substantially increased self-renewal of early lymphoid precursors and lymphomyeloid multipotent progenitors, as well as lymphoid priming that concommitently
reduced myelo-erythroid diferentiation potential of lympho-myeloid multipotent progenitor cells (Fig. 1). In functional assays, BRAFV600E HSCs had a competitive repopulation advantage as compared with normal HSCs, along with substantial lymphoid lineage skewing. Together, these models show that targeting the BRAFV600E mutation to HSCs but not B lineage cells resulted in perturbed hematopoiesis, with many of the hallmark features of HCL. Together with the human data, these animal models strongly support the origin of HCL in cells much earlier than mature B cells. CLINICAL IMPLICATIONS One of the clinical implications of the work by Chung et al. (3) is that continued persistence of ancestral nonleukemic stem cells provides a potential reservoir that could cause disease recurrence even when all the malignant cells are efectively treated. Tus, therapeutic strategies should ensure that both malignant cells and ancestral stem cells are targeted. Te link between BRAFV600E and HCL and HCL-HSC raises the possibility that this disease might be sensitive to the BRAF inhibitor vemurafenib. Te authors found that drug treatment in the BRAFV600E HSC mouse models corrected the disease manifestations. Tey also presented clinical data from a phase 2 study showing that the frequencies of HSCs and progenitors were normalized within 3 months of therapy. Further, there were marked increases in the number of myeloid and erythroid progenitors afer vemurafenib treatment, pointing to a direct link between the BRAFV600E mutation and perturbed myelo-erythroid diferentiation. More genetic analysis will be required to confrm that BRAFV600E-bearing HSCs are eliminated in these patients, but the preliminary results are promising. It remains to be seen whether the new work will yield biomarkers that could be tracked in the context of clinical trials so as to monitor the efects of therapy, not only on the malignant hairy cells, but also on the ancestral cell types that might serve as reservoirs for future recurrence. Te discovery of ancestral cell types buried in clinical samples from AML and now HCL patients should spur the feld to dig for them in other leukemias and lymphomas.
REFERENCES AND NOTES 1. M. S. Lawrence, P. Stojanov, C. H. Mermel, J. T. Robinson, L. A. Garraway, T. R. Golub, M. Meyerson, S. B. Gabriel, E. S. Lander, G. Getz, Discovery and saturation analysis of cancer genes across 21 tumour types. Nature 505, 495–501 (2014). 2. M. Greaves, C. C. Maley, Clonal evolution in cancer. Nature 481, 306–313 (2012). 3. S. S. Chung, E. Kim, J. H. Park, Y. R. Chung, P. Lito, J. TeruyaFeldstein, W. Hu, W. Beguelin, S. Monette, C. Duy, R. Rampal, L. Telis, M. Patel, M. K. Kim, K. Huberman, N. Bouvier, M. F. Berger, A. M. Melnick, N. Rosen, M. S. Tallman, C. Y. Park, O. Abdel-Wahab, Hematopoietic stem cell origin of BRAFV600E mutations in hairy cell leukemia. Sci. Transl. Med. 6, 238ra71 (2014). 4. A. Pandolfi, L. Barreyro, U. Steidl, Concise review: Preleukemic stem cells: Molecular biology and clinical implications of the precursors to leukemia stem cells. Stem Cells Transl. Med. 2, 143–150 (2013). 5. S. Doulatov, F. Notta, E. Laurenti, J. E. Dick, Hematopoiesis: A human perspective. Cell Stem Cell 10, 120–136 (2012). 6. L. I. Shlush, S. Zandi, A. Mitchell, W. C. Chen, J. M. Brandwein, V. Gupta, J. A. Kennedy, A. D. Schimmer, A. C. Schuh, K. W. Yee, J. L. McLeod, M. Doedens, J. J. Medeiros, R. Marke, H. J. Kim, K. Lee, J. D. McPherson, T. J. Hudson, A. M. Brown, F. Yousif, Q. M. Trinh, L. D. Stein, M. D. Minden, J. C. Wang, J. E. Dick, HALT Pan-Leukemia Gene Panel Consortium, Identification of pre-leukaemic haematopoietic stem cells in acute leukaemia. Nature 506, 328–333 (2014). 7. M. R. Corces-Zimmerman, W. J. Hong, I. L. Weissman, B. C. Medeiros, R. Majeti, Preleukemic mutations in human acute myeloid leukemia affect epigenetic regulators and persist in remission. Proc. Natl. Acad. Sci. U.S.A. 111, 2548–2553 (2014). 8. C. Leung-Hagesteijn, N. Erdmann, G. Cheung, J. J. Keats, A. K. Stewart, D. E. Reece, K. C. Chung, R. E. Tiedemann, Xbp1s-negative tumor B cells and pre-plasmablasts mediate therapeutic proteasome inhibitor resistance in multiple myeloma. Cancer Cell 24, 289–304 (2013). 9. J. Zhang, L. Ding, L. Holmfeldt, G. Wu, S. L. Heatley, D. Payne-Turner, J. Easton, X. Chen, J. Wang, M. Rusch, C. Lu, S. C. Chen, L. Wei, J. R. Collins-Underwood, J. Ma, K. G. Roberts, S. B. Pounds, A. Ulyanov, J. Becksfort, P. Gupta, R. Huether, R. W. Kriwacki, M. Parker, D. J. McGoldrick, D. Zhao, D. Alford, S. Espy, K. C. Bobba, G. Song, D. Pei, C. Cheng, S. Roberts, M. I. Barbato, D. Campana, E. CoustanSmith, S. A. Shurtleff, S. C. Raimondi, M. Kleppe, J. Cools, K. A. Shimano, M. L. Hermiston, S. Doulatov, K. Eppert, E. Laurenti, F. Notta, J. E. Dick, G. Basso, S. P. Hunger, M. L. Loh, M. Devidas, B. Wood, S. Winter, K. P. Dunsmore, R. S. Fulton, L. L. Fulton, X. Hong, C. C. Harris, D. J. Dooling, K. Ochoa, K. J. Johnson, J. C. Obenauer, W. E. Evans, C. H. Pui, C. W. Naeve, T. J. Ley, E. R. Mardis, R. K. Wilson, J. R. Downing, C. G. Mullighan, The genetic basis of early T-cell precursor acute lymphoblastic leukaemia. Nature 481, 157–163 (2012). 10. Y. Kikushige, F. Ishikawa, T. Miyamoto, T. Shima, S. Urata, G. Yoshimoto, Y. Mori, T. Iino, T. Yamauchi, T. Eto, H. Niiro, H. Iwasaki, K. Takenaka, K. Akashi, Self-renewing hematopoietic stem cell is the primary target in pathogenesis of human chronic lymphocytic leukemia. Cancer Cell 20, 246–259 (2011). 10.1126/scitranslmed.3009168 Citation: J. E. Dick, Tumor archaeology: Tracking leukemic evolution to its origins. Sci. Transl. Med. 6, 238fs23 (2014).
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was subjected to deep, targeted genetic analysis of the BRAF gene, which demonstrated that the phenotypically normal cells contained the same BRAFV600E mutation that is the hallmark of hairy cells. Sorted HSC fractions from some patient samples possessed a variant allele frequency (VAF) of >5%, indicating marked clonal expansion of the mutant HSCs in HCL. Strictly speaking, fnding the BRAFV600E mutation in phenotypic HSC does not prove that this represents a pre-leukemic ancestral cell type, because cell surface phenotypes may be aberrant in hairy cell leukemia. To address this caveat, three approaches were taken. Additional mutations were detected that were present in all HCL cells, whereas sorted HSCs did not carry the mutations, as would be expected for nonmalignant ancestral cells. Te BRAFV600Ebearing HSCs must have been functionally multipotent because they gave rise to primitive B cell progenitors, myeloid progenitors, and HCL cells in patients, and when transplanted into mice, the cells generated a xenograf composed of phenotypically defned hairy cells and multiple other hematopoietic lineages.
Tumor Archaeology: Tracking Leukemic Evolution to Its Origins John E. Dick Unearthing of the BRAF mutation in self-renewing hematopoietic stem cells reveals an unexpected origin for hairy cell leukemia—a mature B cell malignancy—and a potential new therapeutic target (Chung et al., this issue).
Cancer arises from the multistep accumulation of mutations, and concerted eforts have yielded an unprecedented understanding of genomic lesions in human cancers (1). Trough the application of single-cell genetic analysis and computational algorithms to uncover genetic diversity from whole-genome sequencing of bulk tumor tissue, it has become clear that cancers are composed of multiple genetically distinct subclones. By applying the tools of population genetics, it has been possible to trace the evolutionary history that created this complex clonal architecture (2). However, these approaches examine only malignant cells, leaving unresolved critical questions regarding the earlier premalignant steps in cancer development. What is the identity of the cell of origin? What is the nature and biological consequences of the initiating genetic lesion? What is the order and number of subsequent mutations needed before cells become overtly malignant? In this issue of Science Translational Medicine, new work by Chung et al. points to hematopoietic stem cells (HSCs) as the potential origin for at least some mature B cell malignancies (3). ANCESTRAL STEPPING STONES For most cancer types, the premalignant phase is clinically silent, making it impossible to address the key questions. However, in acute myeloid leukemia (AML), a subset of cases evolve from a preceding clinically apparent clonal hematological disorder such as a myelodysplastic syndrome or a myeloproliferative neoplasm (4). In many of these cases, the lesion that initiates clonal expansion occurs in HSCs in the bone marrow and is present in all descendent cells, including the AML cells that arise afer acquisition of additional mutations in the founding clone. Princess Margaret Cancer Centre, University Health Network and Department of Molecular Genetics, University of Toronto, Ontario M5G2C1, Canada. E-mail: [email protected]
Tese cases of leukemic progression raise the question of whether some ancestral cell types, bearing only the initiating mutations, persist within the hematopoietic tissues (bone marrow and blood) afer overt leukemia has developed. Te evolutionary history of the leukemia would be hidden in such cells, and analysis of their phenotype and mutations would uncover the “stepping stones” of leukemic progression, which could be followed back to the cell of origin. Te plausibility that leukemic blood samples contain nonleukemic cell types that are ancestral to leukemic blasts was strengthened in recent studies of AML that took advantage of great strides made in our ability to isolate, assay, and characterize pure cell populations that represent the entire human hematopoietic stem and progenitor landscape (5). Collectively, these studies found that peripheral blood and bone marrow samples from patients with AML contain hematopoietic stem and progenitor cells as well as mature T and B cells that bear only a single or a subset of the cancer-associated mutations present in the leukemia blasts (6, 7). Despite the acquisition of mutations, these HSCs retain the ability to diferentiate into a full spectrum of myelo-lymphoid lineages (Fig. 1). However, examination of the proportional contribution that HSCs that carry the initiating mutations made to the overall HSC pool indicates that these mutations confer a competitive growth advantage, leading to clonal expansion. Tese studies established that in a high proportion of AML cases, initiating mutations arise in a primitive cell, likely a HSC, to create a pre-leukemic HSC (preLHSC) that clonally expands. AML evolves from this pool of preL-HSCs through the acquisition of additional genetic alterations. THE ORIGIN OF MALIGNANCIES A key principle to be inferred from the studies described above is that initiating mutations must occur in self-renewing cells
to ensure that a clonally expanded population persists and thus allows for the acquisition of subsequent mutations that lead to malignancy. Mutations in non–self-renewing cells would be purged by diferentiation before additional mutations could be acquired. Once myeloid commitment has occurred, no self-renewing cells remain; thus, in AML, initiating mutations must occur in HSCs. However, in normal lymphoid development, many types of progenitor and mature T and B cells possess a substantial capacity for clonal expansion through antigenic stimulation. Indeed, mature memory B cells retain stem cell–like properties to ensure proper immune function upon future antigen exposure. Tus, many cell types throughout lymphoid maturation could act as putative cells of origin for leukemia or lymphoma development. B cell lymphopoiesis is relatively well understood from a cellular and molecular standpoint; there is a highly ordered gain or loss of specifc cell-surface markers that is coupled with specifc immunoglobulin gene rearrangements and transcription factor expression representing specifc stages of development. Tus, it is tempting to infer the identity of the cell of origin by comparing the blocked development that occurs as a result of malignant transformation to a specifc normal developmental stage. For example, if the cellular, molecular, and signaling phenotype of the malignant cells matches that of a mature plasma cell, this would suggest that the cell of origin is also a mature cell. However, this line of reasoning has several faws. Mutations acquired early might lead to clonal expansion but with retention of normal diferentiation. Only mutations acquired at later stages of leukemogenesis may cause a block in diferentiation that leads to malignancy. In this scenario, the phenotype of the cell that acquires the frst mutation will be completely diferent from the phenotype of the malignant cells. Second, not every leukemia cell is equally able to maintain long-term clonal propagation; some have stem cell–like self-renewal properties. Such leukemia-propagating stem cells have cell-surface markers and growth properties that are distinct from those of bulk malignant cells. If such leukemiapropagating cells happen to be rare, they would be missed when studying bulk cell populations. Te properties of such cells might be more closely related to the cell of origin than to the bulk malignant population.
www.ScienceTranslationalMedicine.org 28 May 2014 Vol 6 Issue 238 238fs23
1
Downloaded from on July 3, 2015
FOCUS
Skin Marrow
Hip bone
Normal HSC
B cell expansion Additional mutations
BRAFV600E mutation
HCL-HSC Skewed B cell development
Aberrant self-renewal, clonal expansion
MLP
Granulocytes Dendritic cells/ macrophages CMP Erythrocytes MEP Platelets Reduced myelo-erythroid development GMP
Dendritic T NK cells
MPP
CREDIT: V. ALTOUNIAN/SCIENCE TRANSLATIONAL MEDICINE
Hairy cell
Fig. 1. Backtracking to leukemia’s origin. Acquisition of BRAFV600E mutations within HSCs enhances B cell development and impairs differentiation into other lymphoid and myelo-erythroid blood lineages. The mutated HSCs have increased self-renewal capacity (indicated by thick yellow arrow) resulting in an expanded hairy cell leukemia HSC clone (HCL-HSC) as well as downstream progenitors [multipotential progenitor (MPP) and multilymphoid progenitor (MLP)] that all contain the BRAFV600E mutation. BRAFV600E contributes to enhanced expansion of B cells (indicated by thick black arrows) that then acquire additional mutations, resulting in clinically overt HCL. T, T cell; NK, natural killer cell; CMP, common myeloid progenitor; GMP, granulocyte-macrophage progenitor; MEP, myelo-erythroid precursor.
Indeed, recent evidence suggests that accepted models of development of mature lymphoid malignancies may have been confounded by these issues. For example, recent studies indicate that multiple myeloma, a disease of mature plasma cells, is organized as a hierarchy sustained by rare myelomapropagating cells that are more developmentally immature than the predominant malignant plasma cells; these cells also contribute to proteasome-inhibitor resistance (8).
Genomic analysis of an aggressive subtype of T-ALL (ETP T-ALL) uncovered transcriptional programs and mutations not typically associated with T cell development; rather, mutations were detected in genes that encode proteins that are important for stem cell function, suggesting that cancer-driving mutations originated in a cell type more immature than committed T cells (9). Chronic lymphocytic leukemia (CLL) is a common malignancy characterized by
the clonal expansion of mature CD5+ B cells bearing functional B cell receptors (BCRs) on their surfaces. Antigenic stimulation of these BCRs is typically thought to drive the clonal expansion of a specifc immunoglobulinrearranged cell that might be the cell of origin. However, CLL might be preceded by a B cell lymphocytosis in which multiple subclones are present and only one of these is selected when the disease develops, pointing to an ancestral precursor that had not yet undergone immunoglobulin rearrangement. By sorting bone marrow from CLL patients into various HSC, progenitor, and early B cell lineage populations, provocative evidence has pointed to HSCs and not mature B cells as the origin of the initial cancerdriving mutations (10). Compared with normal HSCs, CLL-HSCs have higher rates of self-renewal and are lymphoid-primed, as evidenced by a higher propensity to generate CD5+ B cells with highly restricted immunoglobulin gene rearrangements typical of the mature CLL cells seen in patients. CLLHSCs also lack translocations found in CLL cells, suggesting that the former cell type is ancestral to the latter. What is missing from these studies is a deep genomic analysis that identifes an initiating mutation that directly implicates HSCs as the cells of origin for malignant CLL B cells. DECONSTRUCTING HAIRY In their study of hairy cell leukemia, Chung et al. now take a substantial leap forward in unveiling HSCs as the cell of origin for at least some mature B cell malignancies (3). Hairy cell leukemia is a chronic lymphoproliferative disorder in which the leukemic hairy cells exhibit classical features of a mature B cell malignancy, including expression of CD19 and surface immunoglobulin and clonal rearrangements of immunoglobulin heavy and light chain genes. In the new work, concerted genome sequencing revealed that virtually all cases of hairy cell leukemia contain a recurrent BRAFV600E mutation. BRAF is a serine/threonine kinase that plays a central role in mitogen-activated protein kinase (MAPK) signaling. Small-molecule inhibitors such as vemurafnib have been developed to block the constitutively active mutant forms of B-RAF. Chung et al. subjected hairy cell leukemia clinical samples to high-resolution fow sorting and isolated populations of phenotypic HSCs, progenitor cells, and mature cells of the hematopoietic lineage. Along with malignant cells, each sorted fraction
www.ScienceTranslationalMedicine.org 28 May 2014 Vol 6 Issue 238 238fs23
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Downloaded from on July 3, 2015
FOCUS
FOCUS
HCL MODELS POINT TO HSC ORIGIN Because it is difcult to carry out mechanistic studies in primary human cells, Chung et al. engineered a number of mouse models in which the BRAFV600E mutation was targeted either to the HSC–progenitor cell compartment or to the B cell lineage alone, with striking results. If BRAFV600E was activated in HSCs or progenitor cells, mice rapidly developed a lethal hematopoietic disorder that was highly similar to HCL and was transplantable into lethally irradiated recipients, which is consistent with a cellautonomous phenotype. In contrast, if the BRAFV600E mutation was targeted to the B cell lineage, no hematopoietic abnormalities were observed for up to 1 year, even though the mutant protein constitutively activated MAPK signaling in B cells. Closer examination of transgenic BRAFV600E mice before the development of overt leukemia demonstrated substantially increased self-renewal of early lymphoid precursors and lymphomyeloid multipotent progenitors, as well as lymphoid priming that concommitently
reduced myelo-erythroid diferentiation potential of lympho-myeloid multipotent progenitor cells (Fig. 1). In functional assays, BRAFV600E HSCs had a competitive repopulation advantage as compared with normal HSCs, along with substantial lymphoid lineage skewing. Together, these models show that targeting the BRAFV600E mutation to HSCs but not B lineage cells resulted in perturbed hematopoiesis, with many of the hallmark features of HCL. Together with the human data, these animal models strongly support the origin of HCL in cells much earlier than mature B cells. CLINICAL IMPLICATIONS One of the clinical implications of the work by Chung et al. (3) is that continued persistence of ancestral nonleukemic stem cells provides a potential reservoir that could cause disease recurrence even when all the malignant cells are efectively treated. Tus, therapeutic strategies should ensure that both malignant cells and ancestral stem cells are targeted. Te link between BRAFV600E and HCL and HCL-HSC raises the possibility that this disease might be sensitive to the BRAF inhibitor vemurafenib. Te authors found that drug treatment in the BRAFV600E HSC mouse models corrected the disease manifestations. Tey also presented clinical data from a phase 2 study showing that the frequencies of HSCs and progenitors were normalized within 3 months of therapy. Further, there were marked increases in the number of myeloid and erythroid progenitors afer vemurafenib treatment, pointing to a direct link between the BRAFV600E mutation and perturbed myelo-erythroid diferentiation. More genetic analysis will be required to confrm that BRAFV600E-bearing HSCs are eliminated in these patients, but the preliminary results are promising. It remains to be seen whether the new work will yield biomarkers that could be tracked in the context of clinical trials so as to monitor the efects of therapy, not only on the malignant hairy cells, but also on the ancestral cell types that might serve as reservoirs for future recurrence. Te discovery of ancestral cell types buried in clinical samples from AML and now HCL patients should spur the feld to dig for them in other leukemias and lymphomas.
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was subjected to deep, targeted genetic analysis of the BRAF gene, which demonstrated that the phenotypically normal cells contained the same BRAFV600E mutation that is the hallmark of hairy cells. Sorted HSC fractions from some patient samples possessed a variant allele frequency (VAF) of >5%, indicating marked clonal expansion of the mutant HSCs in HCL. Strictly speaking, fnding the BRAFV600E mutation in phenotypic HSC does not prove that this represents a pre-leukemic ancestral cell type, because cell surface phenotypes may be aberrant in hairy cell leukemia. To address this caveat, three approaches were taken. Additional mutations were detected that were present in all HCL cells, whereas sorted HSCs did not carry the mutations, as would be expected for nonmalignant ancestral cells. Te BRAFV600Ebearing HSCs must have been functionally multipotent because they gave rise to primitive B cell progenitors, myeloid progenitors, and HCL cells in patients, and when transplanted into mice, the cells generated a xenograf composed of phenotypically defned hairy cells and multiple other hematopoietic lineages.
