From www.bloodjournal.org by guest on September 11, 2016. For personal use only.
l l l LYMPHOID NEOPLASIA
Comment on van Keimpema et al, page 3431
A----------------------------------------------------------------------------------------------------FOXy target in B-cell survival Erin Hertlein
THE OHIO STATE UNIVERSITY
In this issue of Blood, van Keimpema et al present a novel study highlighting the role of FOXP1 in cooperation with nuclear factor kB (NF-kB) in the survival and proliferation of B cells.1
D
iffuse large B-cell lymphoma (DLBCL) accounts for approximately 30% of all lymphomas, with an estimated 10 000 deaths annually in the United States.2,3 It results from uncontrolled growth of B lymphocytes, and it is typically classified into 2 main subgroups based on the resemblance to an activated B cell (ABC) or a germinal center B cell (GCB).4 Agents targeting signaling components of the B-cell receptor (BCR) pathway have produced excellent responses in chronic lymphocytic leukemia and in some types of ABC DLBCL.5 This response is attributed to the fact that these cells exhibit chronic BCR signaling, which causes them to respond to agents that target NF-kB.6 However, there remain few therapeutic options for GCB or ABC lymphomas that do not respond to initial therapy or that eventually relapse. The difficulty in treating DLBCL is in part due to the heterogeneity of the disease, with
several active pathways and various mutations that contribute to pathogenesis. A better understanding of the pathogenic and cellsurvival mechanisms in DLBCL is critical to identify new molecular targets for therapy. Proteins of the forkhead box (FOX) family of transcription factors are involved in cell growth and differentiation, and as such, several members of this family have been implicated in cancer development.7 FOXP1 specifically is a poor prognostic factor in DLBCL8 and may predict transformation to an aggressive lymphoma.9 In their Blood article, van Keimpema et al highlight the antiapoptotic property of FOXP1. They determined relevant FOXP1 target genes by microarray analysis after overexpressing or knocking down FOXP1 in primary B cells from healthy donors as well as DLBCL cell lines, and the predominant pathway identified in these studies is the apoptosis pathway. A set of 7 genes repressed by
The FOXP1 and NF-kB pathways work together to promote the growth of normal primary B cells as well as DLBCL B cells. In DLBCL patients, overall survival can be determined by the expression of a subset of 7 FOXP1 target genes. In the absence of NF-kB signaling, the survival benefit of FOXP1 is lost, indicating the importance of this pathway is most relevant in cells that rely on signaling pathways such as CD40, B-cell receptor (BCR), and Toll-like receptor (TLR) to maintain constitutive NF-kB activation.
3340
FOXP1 was further analyzed in a large set of 498 DLBCL patients on a clinical trial evaluating rituximab plus CHOP, and lower expression of this gene set was found to correlate with shorter overall survival and progression-free survival, which was irrespective of ABC vs GCB subtype. Therefore, lower expression of these genes, presumably due to higher FOXP1 expression, is indicative of a poor prognosis. The authors performed functional studies to corroborate the role in apoptosis, which is indicated by the gene expression studies. They show that overexpression of FOXP1 in primary B cells (in the presence of CD40 ligand stimulation) was able to promote B-cell survival (indicated by decreased caspase-3/7 activation), whereas small interfering RNA knockdown of FOXP1 in DLBCL cell lines increased apoptotic cell death. There was no impact on cellular proliferation (determined by carboxyfluorescein diacetate succinimidyl ester dilution assays) upon knockdown of FOXP1, indicating that the role of FOXP1 is primarily on cell viability. Because the role of FOXP1 was determined in cell lines cultured with CD40L, which can activate NF-kB, the authors went on to determine the contribution of NF-kB to the FOXP1-mediated survival. FOXP1 had no effect in cells without NF-kB activation (provided by either CD40L or constitutively active IKK protein, CA-IKK2). However, coexpression of FOXP1 and CA-IKK2 protein verifies that NF-kB provides a proliferative effect whereas FOXP1 provides an antiapoptotic effect. These 2 parallel pathways work together to maintain the increased number of primary B cells in culture. This also suggests that therapeutically disrupting FOXP1 function may be expected to have a stronger effect in ABC DLBCL, which has been shown to have a strong dependence on NF-kB signaling. In summary, these studies further define the mechanism of FOXP1-mediated B-cell survival and the role of NF-kB. The authors identify a set of 7 FOXP1 target genes that clearly correlate with survival in DLBCL patients and therefore may be useful as a diagnostic tool. Because this work uncovers a clear survival benefit with activation of both FOXP1 and NF-kB together over either pathway alone, this opens the door to therapeutic combination strategies in lymphoma. Conflict-of-interest disclosure: The author declares no competing financial interests. n
BLOOD, 27 NOVEMBER 2014 x VOLUME 124, NUMBER 23
From www.bloodjournal.org by guest on September 11, 2016. For personal use only.
REFERENCES 1. van Keimpema M, Gruneberg ¨ LJ, Mokry M, et al. FOXP1 directly represses transcription of proapoptotic genes and cooperates with NF-kB to promote survival of human B cells. Blood. 2014;124(23):3431-3440.
open-label, phase 2 study [abstract]. Blood. 2012;120(21). Abstract 686. 6. Yang Y, Shaffer AL III, Emre NC, et al. Exploiting synthetic lethality for the therapy of ABC diffuse large B cell lymphoma. Cancer Cell. 2012;21(6):723-737.
2. Abramson JS, Zelenetz AD. Recent advances in the treatment of non-Hodgkin’s lymphomas. J Natl Compr Canc Netw. 2013;11(5 suppl):671-675.
7. Katoh M, Igarashi M, Fukuda H, Nakagama H, Katoh M. Cancer genetics and genomics of human FOX family genes. Cancer Lett. 2013;328(2):198-206.
3. Zelenetz AD, Wierda WG, Abramson JS, et al; National Comprehensive Cancer Network. Non-Hodgkin’s lymphomas, version 1.2013. J Natl Compr Canc Netw. 2013;11(3):257-272, quiz 273.
8. Banham AH, Connors JM, Brown PJ, et al. Expression of the FOXP1 transcription factor is strongly associated with inferior survival in patients with diffuse large B-cell lymphoma. Clin Cancer Res. 2005;11(3):1065-1072.
4. Alizadeh AA, Eisen MB, Davis RE, et al. Distinct types of diffuse large B-cell lymphoma identified by gene expression profiling. Nature. 2000;403(6769):503-511. 5. Wilson WH, Gerecitano JF, Goy A, et al. The Bruton’s tyrosine kinase (BTK) inhibitor, ibrutinib (pci-32765), has preferential activity in the ABC subtype of relapsed/refractory de novo diffuse large B-cell lymphoma (DLBCL): interim results of a multicenter,
9. Sagaert X, de Paepe P, Libbrecht L, et al. Forkhead box protein P1 expression in mucosa-associated lymphoid tissue lymphomas predicts poor prognosis and transformation to diffuse large B-cell lymphoma. J Clin Oncol. 2006;24(16):2490-2497. © 2014 by The American Society of Hematology
l l l MYELOID NEOPLASIA
Comment on Schlenk et al, page 3441
Allelic ratio: a marker of clonal dominance ----------------------------------------------------------------------------------------------------Soheil Meshinchi
FRED HUTCHINSON CANCER RESEARCH INSTITUTE
In this issue of Blood, Schlenk et al provide compelling data on the prognostic implications of FMS-like tyrosine kinase 3 (FLT3)/internal tandem duplication allelic ratio (ITD-AR), as well as its impact on response to allogeneic stem cell transplantation.1
T
hey provide detailed analysis of data from a large cohort of patients with FLT3-ITD treated in 4 multicenter German-Austrian Acute Myeloid Leukemia (AML) Study Group (AMLSG) trials and correlate the variation in ITD-AR with response to induction chemotherapy and clinical outcome. Given the significant variation in ITD-AR and lack of a preestablished biological threshold, the authors establish a statistically defined cut-point for clinically meaningful ITD-AR of 0.51 for further correlation with clinical outcome. It is important to note that this ITD-AR is similar to those previously defined clinically defined thresholds.2 They demonstrate that those with high ITD-AR have a significantly worse complete remission (CR) rate and correspondingly poor survival and relapse. They further demonstrate that the adverse outcome of high ITD-AR is abrogated by stem cell transplantation. In contrast to patients with high ITD-AR, there appears to be no distinction between
those with low ITD-AR and those without FLT3-ITD (FLT3 wild type [FLT3/WT]) in terms of overall outcome or response to hematopoietic stem cell transplantation (HSCT). To discuss the role of varying ITD-AR in AML, one must address the underlying mechanism for such a wide variation in AR (ranging from 0.01 to .100), which is the ultimate representation of the enormous complexity and genomic heterogeneity of FLT3-ITD–positive AML, as multiple factors contribute to this variation in ITD-AR. The most obvious of these factors is that ITD-AR is a representation of the state of clonal dominance (or lack thereof) of the mutation, where in those with high ITD-AR, FLT3-ITD is the dominant lesion and is present in the majority or all of the leukemic cells. In contrast, in those with low ITD-AR, FLT3-ITD is present in a minor subclone within the bulk leukemic population. Single cell genotyping of leukemic cells from patients with varying ITD-AR established
BLOOD, 27 NOVEMBER 2014 x VOLUME 124, NUMBER 23
the genomic heterogeneity in FLT3-ITD AML, demonstrating that FLT3-ITD may be present in a major (high ITD-AR) or minor (low ITD-AR) subset of leukemic cells, and defined an association of ITD-AR with the proportion of individual cells that harbor FLT3-ITD and contribute to the final ITD-AR value.3 ITD-AR is further impacted by the associated copy-neutral loss of heterozygosity (LOH) of chromosome 13q (13q CN-LOH) in patients with FLT3-ITD. This genomic variant is the result of a homologous recombination-mediated process where the WT allele is replaced by the allele with FLT3-ITD, resulting in loss of heterozygosity (without copy number loss), leading to evolution of homozygous ITD. Single cell genotyping further demonstrated that in patients with FLT3-ITD, leukemic cells are composed of a mixture of homozygous FLT3-ITD, heterozygous FLT3-ITD, and FLT3/WT, with a final value of ITD-AR determined by the proportion of each genotypic subset.3 The fact that 13q CN-LOH is uniquely observed in the setting of FLT3-ITD may suggest a causal association between FLT3-ITD and evolution of this unique genomic event. Ultimately, ITD-AR represents the extent of the clonal dominance of FLT3-ITD (with contribution of 13q CN-LOH), where in those with high ITD-AR, FLT3-ITD is present in the majority of the leukemic cells and likely the dominant and driving genomic event. In contrast, in those with low ITD-AR, where FLT3-ITD is present in the minority of leukemic cells, FLT3-ITD is unlikely to be the driving event and contribute to leukemic relapse. The clinical impact of AR has been observed in other mutations by demonstration of the association of the AR of KIT and CBL mutations with outcome.4 These data suggest that we may need to alter our approach to evaluation of diagnostic mutations in AML and determine not only what the mutation is, but whether the mutation is a dominant, driving lesion or a minor variant. An additional layer of data that substantiates the significance of mutation burden at diagnosis is the fact that the majority of diagnostic mutations with low allele fraction (low AR) are not detected at relapse, thus questioning their clinical significance.5 Demonstration of the clinical significance of FLT3-ITD led to the evaluation of HSCT in this high-risk cohort. Initial studies demonstrated that HSCT abrogates the
3341
From www.bloodjournal.org by guest on September 11, 2016. For personal use only.
2014 124: 3340-3341 doi:10.1182/blood-2014-10-605014
A FOXy target in B-cell survival Erin Hertlein
Updated information and services can be found at: http://www.bloodjournal.org/content/124/23/3340.full.html Articles on similar topics can be found in the following Blood collections Free Research Articles (4041 articles) Information about reproducing this article in parts or in its entirety may be found online at: http://www.bloodjournal.org/site/misc/rights.xhtml#repub_requests Information about ordering reprints may be found online at: http://www.bloodjournal.org/site/misc/rights.xhtml#reprints Information about subscriptions and ASH membership may be found online at: http://www.bloodjournal.org/site/subscriptions/index.xhtml
Blood (print ISSN 0006-4971, online ISSN 1528-0020), is published weekly by the American Society of Hematology, 2021 L St, NW, Suite 900, Washington DC 20036. Copyright 2011 by The American Society of Hematology; all rights reserved.
l l l LYMPHOID NEOPLASIA
Comment on van Keimpema et al, page 3431
A----------------------------------------------------------------------------------------------------FOXy target in B-cell survival Erin Hertlein
THE OHIO STATE UNIVERSITY
In this issue of Blood, van Keimpema et al present a novel study highlighting the role of FOXP1 in cooperation with nuclear factor kB (NF-kB) in the survival and proliferation of B cells.1
D
iffuse large B-cell lymphoma (DLBCL) accounts for approximately 30% of all lymphomas, with an estimated 10 000 deaths annually in the United States.2,3 It results from uncontrolled growth of B lymphocytes, and it is typically classified into 2 main subgroups based on the resemblance to an activated B cell (ABC) or a germinal center B cell (GCB).4 Agents targeting signaling components of the B-cell receptor (BCR) pathway have produced excellent responses in chronic lymphocytic leukemia and in some types of ABC DLBCL.5 This response is attributed to the fact that these cells exhibit chronic BCR signaling, which causes them to respond to agents that target NF-kB.6 However, there remain few therapeutic options for GCB or ABC lymphomas that do not respond to initial therapy or that eventually relapse. The difficulty in treating DLBCL is in part due to the heterogeneity of the disease, with
several active pathways and various mutations that contribute to pathogenesis. A better understanding of the pathogenic and cellsurvival mechanisms in DLBCL is critical to identify new molecular targets for therapy. Proteins of the forkhead box (FOX) family of transcription factors are involved in cell growth and differentiation, and as such, several members of this family have been implicated in cancer development.7 FOXP1 specifically is a poor prognostic factor in DLBCL8 and may predict transformation to an aggressive lymphoma.9 In their Blood article, van Keimpema et al highlight the antiapoptotic property of FOXP1. They determined relevant FOXP1 target genes by microarray analysis after overexpressing or knocking down FOXP1 in primary B cells from healthy donors as well as DLBCL cell lines, and the predominant pathway identified in these studies is the apoptosis pathway. A set of 7 genes repressed by
The FOXP1 and NF-kB pathways work together to promote the growth of normal primary B cells as well as DLBCL B cells. In DLBCL patients, overall survival can be determined by the expression of a subset of 7 FOXP1 target genes. In the absence of NF-kB signaling, the survival benefit of FOXP1 is lost, indicating the importance of this pathway is most relevant in cells that rely on signaling pathways such as CD40, B-cell receptor (BCR), and Toll-like receptor (TLR) to maintain constitutive NF-kB activation.
3340
FOXP1 was further analyzed in a large set of 498 DLBCL patients on a clinical trial evaluating rituximab plus CHOP, and lower expression of this gene set was found to correlate with shorter overall survival and progression-free survival, which was irrespective of ABC vs GCB subtype. Therefore, lower expression of these genes, presumably due to higher FOXP1 expression, is indicative of a poor prognosis. The authors performed functional studies to corroborate the role in apoptosis, which is indicated by the gene expression studies. They show that overexpression of FOXP1 in primary B cells (in the presence of CD40 ligand stimulation) was able to promote B-cell survival (indicated by decreased caspase-3/7 activation), whereas small interfering RNA knockdown of FOXP1 in DLBCL cell lines increased apoptotic cell death. There was no impact on cellular proliferation (determined by carboxyfluorescein diacetate succinimidyl ester dilution assays) upon knockdown of FOXP1, indicating that the role of FOXP1 is primarily on cell viability. Because the role of FOXP1 was determined in cell lines cultured with CD40L, which can activate NF-kB, the authors went on to determine the contribution of NF-kB to the FOXP1-mediated survival. FOXP1 had no effect in cells without NF-kB activation (provided by either CD40L or constitutively active IKK protein, CA-IKK2). However, coexpression of FOXP1 and CA-IKK2 protein verifies that NF-kB provides a proliferative effect whereas FOXP1 provides an antiapoptotic effect. These 2 parallel pathways work together to maintain the increased number of primary B cells in culture. This also suggests that therapeutically disrupting FOXP1 function may be expected to have a stronger effect in ABC DLBCL, which has been shown to have a strong dependence on NF-kB signaling. In summary, these studies further define the mechanism of FOXP1-mediated B-cell survival and the role of NF-kB. The authors identify a set of 7 FOXP1 target genes that clearly correlate with survival in DLBCL patients and therefore may be useful as a diagnostic tool. Because this work uncovers a clear survival benefit with activation of both FOXP1 and NF-kB together over either pathway alone, this opens the door to therapeutic combination strategies in lymphoma. Conflict-of-interest disclosure: The author declares no competing financial interests. n
BLOOD, 27 NOVEMBER 2014 x VOLUME 124, NUMBER 23
From www.bloodjournal.org by guest on September 11, 2016. For personal use only.
REFERENCES 1. van Keimpema M, Gruneberg ¨ LJ, Mokry M, et al. FOXP1 directly represses transcription of proapoptotic genes and cooperates with NF-kB to promote survival of human B cells. Blood. 2014;124(23):3431-3440.
open-label, phase 2 study [abstract]. Blood. 2012;120(21). Abstract 686. 6. Yang Y, Shaffer AL III, Emre NC, et al. Exploiting synthetic lethality for the therapy of ABC diffuse large B cell lymphoma. Cancer Cell. 2012;21(6):723-737.
2. Abramson JS, Zelenetz AD. Recent advances in the treatment of non-Hodgkin’s lymphomas. J Natl Compr Canc Netw. 2013;11(5 suppl):671-675.
7. Katoh M, Igarashi M, Fukuda H, Nakagama H, Katoh M. Cancer genetics and genomics of human FOX family genes. Cancer Lett. 2013;328(2):198-206.
3. Zelenetz AD, Wierda WG, Abramson JS, et al; National Comprehensive Cancer Network. Non-Hodgkin’s lymphomas, version 1.2013. J Natl Compr Canc Netw. 2013;11(3):257-272, quiz 273.
8. Banham AH, Connors JM, Brown PJ, et al. Expression of the FOXP1 transcription factor is strongly associated with inferior survival in patients with diffuse large B-cell lymphoma. Clin Cancer Res. 2005;11(3):1065-1072.
4. Alizadeh AA, Eisen MB, Davis RE, et al. Distinct types of diffuse large B-cell lymphoma identified by gene expression profiling. Nature. 2000;403(6769):503-511. 5. Wilson WH, Gerecitano JF, Goy A, et al. The Bruton’s tyrosine kinase (BTK) inhibitor, ibrutinib (pci-32765), has preferential activity in the ABC subtype of relapsed/refractory de novo diffuse large B-cell lymphoma (DLBCL): interim results of a multicenter,
9. Sagaert X, de Paepe P, Libbrecht L, et al. Forkhead box protein P1 expression in mucosa-associated lymphoid tissue lymphomas predicts poor prognosis and transformation to diffuse large B-cell lymphoma. J Clin Oncol. 2006;24(16):2490-2497. © 2014 by The American Society of Hematology
l l l MYELOID NEOPLASIA
Comment on Schlenk et al, page 3441
Allelic ratio: a marker of clonal dominance ----------------------------------------------------------------------------------------------------Soheil Meshinchi
FRED HUTCHINSON CANCER RESEARCH INSTITUTE
In this issue of Blood, Schlenk et al provide compelling data on the prognostic implications of FMS-like tyrosine kinase 3 (FLT3)/internal tandem duplication allelic ratio (ITD-AR), as well as its impact on response to allogeneic stem cell transplantation.1
T
hey provide detailed analysis of data from a large cohort of patients with FLT3-ITD treated in 4 multicenter German-Austrian Acute Myeloid Leukemia (AML) Study Group (AMLSG) trials and correlate the variation in ITD-AR with response to induction chemotherapy and clinical outcome. Given the significant variation in ITD-AR and lack of a preestablished biological threshold, the authors establish a statistically defined cut-point for clinically meaningful ITD-AR of 0.51 for further correlation with clinical outcome. It is important to note that this ITD-AR is similar to those previously defined clinically defined thresholds.2 They demonstrate that those with high ITD-AR have a significantly worse complete remission (CR) rate and correspondingly poor survival and relapse. They further demonstrate that the adverse outcome of high ITD-AR is abrogated by stem cell transplantation. In contrast to patients with high ITD-AR, there appears to be no distinction between
those with low ITD-AR and those without FLT3-ITD (FLT3 wild type [FLT3/WT]) in terms of overall outcome or response to hematopoietic stem cell transplantation (HSCT). To discuss the role of varying ITD-AR in AML, one must address the underlying mechanism for such a wide variation in AR (ranging from 0.01 to .100), which is the ultimate representation of the enormous complexity and genomic heterogeneity of FLT3-ITD–positive AML, as multiple factors contribute to this variation in ITD-AR. The most obvious of these factors is that ITD-AR is a representation of the state of clonal dominance (or lack thereof) of the mutation, where in those with high ITD-AR, FLT3-ITD is the dominant lesion and is present in the majority or all of the leukemic cells. In contrast, in those with low ITD-AR, FLT3-ITD is present in a minor subclone within the bulk leukemic population. Single cell genotyping of leukemic cells from patients with varying ITD-AR established
BLOOD, 27 NOVEMBER 2014 x VOLUME 124, NUMBER 23
the genomic heterogeneity in FLT3-ITD AML, demonstrating that FLT3-ITD may be present in a major (high ITD-AR) or minor (low ITD-AR) subset of leukemic cells, and defined an association of ITD-AR with the proportion of individual cells that harbor FLT3-ITD and contribute to the final ITD-AR value.3 ITD-AR is further impacted by the associated copy-neutral loss of heterozygosity (LOH) of chromosome 13q (13q CN-LOH) in patients with FLT3-ITD. This genomic variant is the result of a homologous recombination-mediated process where the WT allele is replaced by the allele with FLT3-ITD, resulting in loss of heterozygosity (without copy number loss), leading to evolution of homozygous ITD. Single cell genotyping further demonstrated that in patients with FLT3-ITD, leukemic cells are composed of a mixture of homozygous FLT3-ITD, heterozygous FLT3-ITD, and FLT3/WT, with a final value of ITD-AR determined by the proportion of each genotypic subset.3 The fact that 13q CN-LOH is uniquely observed in the setting of FLT3-ITD may suggest a causal association between FLT3-ITD and evolution of this unique genomic event. Ultimately, ITD-AR represents the extent of the clonal dominance of FLT3-ITD (with contribution of 13q CN-LOH), where in those with high ITD-AR, FLT3-ITD is present in the majority of the leukemic cells and likely the dominant and driving genomic event. In contrast, in those with low ITD-AR, where FLT3-ITD is present in the minority of leukemic cells, FLT3-ITD is unlikely to be the driving event and contribute to leukemic relapse. The clinical impact of AR has been observed in other mutations by demonstration of the association of the AR of KIT and CBL mutations with outcome.4 These data suggest that we may need to alter our approach to evaluation of diagnostic mutations in AML and determine not only what the mutation is, but whether the mutation is a dominant, driving lesion or a minor variant. An additional layer of data that substantiates the significance of mutation burden at diagnosis is the fact that the majority of diagnostic mutations with low allele fraction (low AR) are not detected at relapse, thus questioning their clinical significance.5 Demonstration of the clinical significance of FLT3-ITD led to the evaluation of HSCT in this high-risk cohort. Initial studies demonstrated that HSCT abrogates the
3341
From www.bloodjournal.org by guest on September 11, 2016. For personal use only.
2014 124: 3340-3341 doi:10.1182/blood-2014-10-605014
A FOXy target in B-cell survival Erin Hertlein
Updated information and services can be found at: http://www.bloodjournal.org/content/124/23/3340.full.html Articles on similar topics can be found in the following Blood collections Free Research Articles (4041 articles) Information about reproducing this article in parts or in its entirety may be found online at: http://www.bloodjournal.org/site/misc/rights.xhtml#repub_requests Information about ordering reprints may be found online at: http://www.bloodjournal.org/site/misc/rights.xhtml#reprints Information about subscriptions and ASH membership may be found online at: http://www.bloodjournal.org/site/subscriptions/index.xhtml
Blood (print ISSN 0006-4971, online ISSN 1528-0020), is published weekly by the American Society of Hematology, 2021 L St, NW, Suite 900, Washington DC 20036. Copyright 2011 by The American Society of Hematology; all rights reserved.
