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Epigenetics Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/kepi20
Dysregulated Class I histone deacetylases are indicators of poor prognosis in multiple myeloma a
b
b
a
abc
Sridurga Mithraprabhu , Anna Kalff , Annie Chow , Tiffany Khong & Andrew Spencer a
Myeloma Research Group; Division of Blood Cancers; Australian Center for Blood Diseases; Alfred Hospital; Monash University; Melbourne, Australia b
Malignant Hematology and Stem Cell Transplantation; Alfred Hospital; Melbourne, Australia
c
Department of Clinical Hematology; Monash University; Clayton, Australia Accepted author version posted online: 06 Dec 2014.Published online: 18 Dec 2014.
Click for updates To cite this article: Sridurga Mithraprabhu, Anna Kalff, Annie Chow, Tiffany Khong & Andrew Spencer (2014) Dysregulated Class I histone deacetylases are indicators of poor prognosis in multiple myeloma, Epigenetics, 9:11, 1511-1520, DOI: 10.4161/15592294.2014.983367 To link to this article: http://dx.doi.org/10.4161/15592294.2014.983367
PLEASE SCROLL DOWN FOR ARTICLE Taylor & Francis makes every effort to ensure the accuracy of all the information (the “Content”) contained in the publications on our platform. However, Taylor & Francis, our agents, and our licensors make no representations or warranties whatsoever as to the accuracy, completeness, or suitability for any purpose of the Content. Any opinions and views expressed in this publication are the opinions and views of the authors, and are not the views of or endorsed by Taylor & Francis. The accuracy of the Content should not be relied upon and should be independently verified with primary sources of information. Taylor and Francis shall not be liable for any losses, actions, claims, proceedings, demands, costs, expenses, damages, and other liabilities whatsoever or howsoever caused arising directly or indirectly in connection with, in relation to or arising out of the use of the Content. This article may be used for research, teaching, and private study purposes. Any substantial or systematic reproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in any form to anyone is expressly forbidden. Terms & Conditions of access and use can be found at http:// www.tandfonline.com/page/terms-and-conditions
RESEARCH PAPER Epigenetics 9:11, 1511--1520; November 2014; © 2014 Taylor & Francis Group, LLC
Dysregulated Class I histone deacetylases are indicators of poor prognosis in multiple myeloma Sridurga Mithraprabhu1, Anna Kalff2, Annie Chow2, Tiffany Khong1, and Andrew Spencer1,2,3,* 1
Myeloma Research Group; Division of Blood Cancers; Australian Center for Blood Diseases; Alfred Hospital; Monash University; Melbourne, Australia; 2Malignant Hematology and Stem Cell Transplantation; Alfred Hospital; Melbourne, Australia; 3Department of Clinical Hematology; Monash University; Clayton, Australia
Keywords: Class I HDAC, HDAC expression, hematological malignancy, histone deacetylases, multiple myeloma, prognosis
Downloaded by [Tulane University] at 22:10 30 January 2015
Abbreviations:
Histone deacetylases (HDAC) control gene expression through their ability to acetylate proteins, thereby influencing a diverse range of cellular functions. Class I HDAC (HDAC1–3 and 8) and HDAC6 are predominantly upregulated in malignancies and their altered expression in some cancers has a significant prognostic implication. The expression and prognostic consequence of dysregulated Class I HDAC and HDAC6, key players in multiple myeloma (MM), are unknown. This study hypothesized that HDAC are dysregulated in MM and patients with high expression have significantly poorer prognostic outcomes. Quantitative PCR for 11 HDAC (Class I, II, and IV) was performed in genetically heterogeneous human myeloma cell lines (HMCL) and primary MM and compared to normal plasma cells (PC). In HMCL, HDAC1–3 and 8 (Class I), and HDAC5 and HDAC10 (Class II) were significantly upregulated compared to normal PC. In primary MM, the median expression level of all of the HDAC, except HDAC1 and HDAC11, were elevated when compared to normal PC. Patients with higher levels of HDAC1–3, HDAC4, HDAC6, and HDAC11 transcripts demonstrated a significantly shorter progression-free survival (PFS). Immunohistochemical staining for HDAC1 and HDAC6 on bone marrow trephines from a uniformly treated cohort of transplant eligible MM patients revealed that HDAC1 protein was detectable in most patients and that higher levels of MM cell HDAC1 protein expression (90 % versus 20 % MM cell positivity) correlated with both shorter PFS (P D 0 .07) and shorter overall survival (P D 0 .003). Conversely, while the majority of patients expressed HDAC6, there was no correlation between HDAC6 levels and patient outcome. Together, these results indicate that overexpression of Class I HDAC, particularly HDAC1, is associated with poor prognosis in MM.
Introduction Multiple myeloma (MM) is an incurable clonal B-cell malignant neoplasm characterized by de novo drug resistance and complex cytogenetic abnormalities that are associated with unique clinical and prognostic implications.1-5 In contrast to the genomic abnormalities, limited information is known about the role of the epigenome in MM pathogenesis and maintenance. Epigenetic modifications, such as DNA methylation and histone acetylation of structurally intact genes have been recognized as critical facets of cancer pathogenesis and maintenance.6 Acetylation is modulated by the dynamic and antagonistic action of 2 classes of enzymes, histone deacetylases (HDAC) and histone acetyltransferases (HAT), wherein HDAC catalyze the removal of acetyl groups and HAT acetylate the N-terminal lysine residues. HDACs are a highly conserved group of enzymes currently consisting of 18 genes grouped into 4 classes, based on their homology to yeast orthologues. Class I (HDAC1–3 and 8), Class IIA (HDAC4, 5, 7, and 9), Class IIB (HDAC6 and 10) and Class IV
(HDAC11) require zinc for catalyzing deacetylase activity, and Class III (Sirtuins 1–7) utilize nicotine adenine dinucleotide (NADC) for their catalytic mechanisms.7,8 HDACs orchestrate a myriad of cellular functions, including proliferation, differentiation, and apoptosis, through the deacetylation of histones and non-histone proteins. Dysregulation of HDAC expression, predominantly overexpression, has been observed in a number of malignancies.9 Class I HDAC expression, in particular, is known to be increased in a number of cancers, including gastric, prostate, colon, breast, renal, and cervical.10-16 Specific to hematological malignancies, dysregulated HDAC expression has been reported in peripheral T-cell lymphomas (PTCL), cutaneous T-cell lymphomas (CTCL), diffuse large B-cell lymphomas (DLBCL), pediatric acute lymphoblastic leukemia (ALL), and myeloproliferative neoplasms.17-20 In all instances, the expression of one or more of Class I HDACs was increased. Prognostic correlates of upregulated HDAC expression are, however, more complex and appear to be highly context-dependent, with the majority of studies
*Correspondence to: Andrew Spencer; Email: [email protected] Submitted: 08/14/2014; Revised: 09/28/2014; Accepted: 10/03/2014 http://dx.doi.org/10.4161/15592294.2014.983367
www.landesbioscience.com
Epigenetics
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Downloaded by [Tulane University] at 22:10 30 January 2015
demonstrating a worse prognosis with higher levels of HDAC1 and/or HDAC2 expression.10-14,21 The opposite effect was seen in breast cancer (HDAC1), ALL, and chronic lymphocytic leukemia (CLL; HDAC3), in which overexpression was a favorable prognostic indicator.16,22,23 HDAC6 has also been studied in cancers because of its to ability to orchestrate a variety of cellular processes that are crucial for cancer pathogenesis.24 Overexpression of HDAC6 has been demonstrated in hepatocellular carcinomas,25 CTCL,17 ALL, 22 and breast cancers.26,27 Moreover, in contrast to increased Class I HDAC expression, which for the most part is an indicator of inferior survival, overexpression of HDAC6 has been largely associated with both improved overall (OS) and progression-free survival (PFS), including in studies of CTCL, 17 breast cancer,26,27 lung cancer,28 DLBCL, 20 and CLL.23 Conversely, a small number of studies have demonstrated that increased HDAC6 expression (breast carcinomas and PTCL) is a negative prognostic factor.15,20 In MM, neither the pattern of expression of HDAC nor any potential association with prognosis has been systematically studied. In vitro HDAC6 appears to be a key modulator of MM cell survival, with studies showing the efficacy of HDAC6-selective inhibitors to induce cell death in MM cells.29-31 Similarly, Class I HDAC also play a critical role in MM cell survival, with evidence showing that inhibition of Class I HDAC induces MM cell death and that inhibition of Class I HDAC is superior to HDAC6 inhibition alone in promoting MM cell apoptosis.32,33 Dysregulation of HDAC may also to contribute to MM drug resistance, with overexpression of HDAC1 possibly conferring resistance to bortezomib-induced apoptosis. This resistance is reversed by the simultaneous addition of FK228, a Class I HDAC inhibitor (HDACi).33 Given the role HDACs play in tumor cell survival and drug resistance and the emergence of HDACi as a potentially available class of anti-MM therapeutics, characterizing the patterns of HDAC expression in MM, and any correlation with patient outcome, is of interest.
Results HDAC levels in HMCL are significantly elevated The levels of expression of HDAC1–11 were determined by qRT-PCR in a genetically heterogeneous panel (n D 14 ) of HMCL and compared to the levels of expression in a panel of normal PC (n D 9 ). All the HDAC transcripts were detected in the tested HMCL, with HDAC11 showing the highest expression and the most variability (mean § SEM: 21.29 § 13.66), followed by HDAC1 (5.89 § 1.03), and HDAC5 (4.96 § 0.56) (Fig. 1). The mean values represent the relative expression levels of the particular gene in HMCL compared to normal PC, which have been normalized to one. Moreover, all Class I HDAC isoforms (HDAC1, 2, 3, and 8), HDAC5, and HDAC10 were significantly elevated in HMCL when compared to normal PC (Fig. 1). To determine if HDAC expression showed any correlation with recognized recurring cytogenetic abnormalities, the HMCL
1512
were grouped based on their translocation status and expression levels of HDAC in these groups were compared. Primary IgH translocations in MM are represented predominantly by 4 recurring abnormalities: 4p16 [t(4;14); FGFR3 and MMSET], 11q13 [t(11;14); CYCLIN D1], 6p21 [t(6;14); CYCLIN D3] and 16q23 [t(14;16); C-MAF]. Among the HMCL chosen for this study, 5/14 harbor t(4;14) (OPM-2, NCI-H929, LP-1, KMS18, and JIM-1), 4/14 have t(11;14) (KMS-12BM, KMS-12PE, XG-1, and U266), 3/14 have t(14;16) (OCI-MY1, RPMI-8226, and ANBL-6), and 2/14 have both t(4;14) and t(14;16) (KMS11 and KMS-26).34-38 KMS-11 and KMS-26, which have more than one primary translocation, were not used for analysis. The t (4;14) HMCL had the highest number of HDAC that were overexpressed (8/11; 72.72%) when compared to normal PC, with all of Class I HDAC, HDAC5, HDAC7, HDAC10, and HDAC11 significantly elevated (Table 1). Class I HDAC, HDAC5, and HDAC7 were also significantly elevated in t(11;14) HMCL (6/11; 54.54%), while t(14;16) HMCL showed overexpression of HDAC1, HDAC2, HDAC3, HDAC5, and HDAC10 and downregulation of HDAC6 (6/11; 54.54%). Secondary translocations, which are more complex and associated with tumor progression, are also prevalent in HMCL, most notably involving 8q24 (c-myc). Eleven of the HMCL used in this study have a translocation involving the c-myc locus (the exceptions being U266, ANBL-6, and JIM-1).39,40 The expression of the majority of the HDAC tested (7/11; 63.63%) was also significantly elevated in these HMCL (Table 1). Notably, HDAC1–3 were elevated in all HMCL irrespective of the translocations. These results demonstrate that HDAC are broadly dysregulated in MM with the dysregulation of the Class I HDACs being most prominent, but with no apparent correlation between the pattern of HDAC dysregulation and any particular karyotypic subsets of MM. HDAC are dysregulated in primary MM, with higher expression correlating to significantly shorter survival To determine if HDAC expression is abnormal in primary MM compared to normal plasma cells cohorts of purified MM tumor cells from both newly diagnosed and relapsed MM patients were evaluated. Table 2 illustrates the patient characteristics of the studied MM population. Analyses of gene expression levels of HDAC in primary MM cells (n D 55 ; clinical details available for n D 49 ) and normal PC (n D 9 ) revealed that the median levels of expression of all HDAC, with the exception of HDAC1, HDAC4, and HDAC11, were significantly elevated in MM, compared to normal PC, in which expression levels were normalized to one for that particular gene (Fig. 2). Median HDAC4 expression levels were reduced in comparison to normal PC. HDAC9 had the highest fold change in median expression levels when compared to normal PC (median 3.31) followed by HDAC3 (median 2.23) and HDAC7 (median: 2.20). The potential prognostic significance of dysregulated HDAC expression was then analyzed. The overall survival (OS – from diagnosis to time of death) and progression-free survival (PFS – from diagnosis to time of progression or death) in MM patients with high expression levels ( 75th centile) compared to low levels ( 75th
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centile) were derived utilizing Kaplan-Meier survival plots. Of the n D 55 MM samples used for qRT-PCR, 37 had information available to determine OS and PFS. Although the median level of expression of HDAC1 in MM patients was not significantly altered compared to normal PC, those patients with elevated HDAC1 expression had shorter PFS compared to patients with low expression (median PFS; High 586 vs Low 1050; P D 0 .0021; Fig. 3A). Similarly, MM patients with elevated HDAC2, HDAC4, HDAC6, and HDAC11 also showed shorter PFS compared to patients with lower expression levels (Fig. 3B-E) indicating that overexpression of HDAC is a predictor for poor prognosis.
Figure 1. HDAC genes are overexpressed in human myeloma cell lines when compared to normal plasma cells Expression levels of HDAC in 14 human myeloma cell lines (HMCL) were compared to normal plasma cells (normal; n D 9 ). Class I HDAC (HDAC1, HDAC2, HDAC3, and HDAC8) were all significantly overexpressed. Among the class II HDAC, HDAC5 and HDAC10 were significantly overexpressed. Data are presented as mean§SEM. Significant differences were calculated with t-test utilizing GraphPad Prism 5.0d. p-values are indicated.
Increased HDAC1 protein expression correlates with poor overall and progression-free survival The qRT-PCR data from the primary MM cohort demonstrated that overexpression of Class I HDAC genes is significantly correlated with a poorer prognosis. To validate our findings at the protein level, a preliminary analysis of HDAC1–3 and HDAC6 protein expression in BM core biopsies from MM patients was first performed to determine the feasibility of such an approach. HDAC2 and HDAC3 protein expression was ubiquitous, being seen in all MM cells and in a majority of non-MM
cells (data not shown); therefore, it was considered that a correlative analysis of HDAC2 and HDAC3 protein expression would be uninterpretable. In contrast, HDAC1 and HDAC6 expression was more MM cell-restricted, allowing MM cell specific determination to be undertaken. Consequently, validation via analysis of HDAC1 and HDAC6 MM cell protein expression was undertaken utilizing a second independent cohort of 97 uniformly treated transplant-eligible MM patients. Of the 97 tested patients, 79 patients showed assessable HDAC1 nuclear staining of MM cells and, within this cohort, 73.4% (58/79) of patients showed 50 % staining (Table 3). Representative images showing 20% HDAC1 staining and a second specimen with 90%
Table 1. HDAC1-2 (Class I HDAC) are overexpressed in all HMCL Primary translocations Gene HDAC1 HDAC2 HDAC3 HDAC8 HDAC5 HDAC7 HDAC6 HDAC10 HDAC11
Secondary translocations
Normal
t(4;14) (FGFR3 and MMSET)
t(11;14) (CYCLIN D1)
t(14;16) (C-MAF)
8q24 (c-MYC)
1.15§0.25 1.18§0.26 1.24§0.29 1.21§0.24 1.36§0.32 1.30§0.33 1.21§0.81 1.12§0.17 2.59§1.06
5.75§0.80**** 4.83§0.48**** 3.44§0.76** 2.03§0.12** 5.41§0.70**** 2.57§0.31** 1.57§0.26 2.76§0.26**** 54.10§26.85*
7.73§2.04**** 5.13§0.88**** 4.00§0.71*** 2.29§0.36* 6.23§1.24*** 2.53§0.20* 2.70§0.93 1.47§0.34 7.85§2.73
3.17§0.41*** 2.21§0.31** 2.65§0.23** 1.66§0.17 3.15§0.54** 1.61§0.17 0.69§0.08* 2.25§0.36* 1.77§0/36
6.67§0.95**** 4.33§0.39**** 3.68§0.44**** 2.29§0.15*** 5.32§0.57**** 2.68§0.31** 1.82§0.37 2.53§0.22** 9.93§3.37
HMCLs were grouped based on their translocation status [t(4;14), t(11;14), t(14;16) and translocations involving c-myc locus – 8q24] and expression levels of HDAC were compared to those in normal plasma cells. HMCL with t(4;14) had significantly higher levels of HDAC (8/11) followed by translocations involving c-myc locus (7/11). HMCLs with t(11;14) and t(14;16) had 6/11 HDACs significantly dysregulated. Significant differences were observed using t-test and GraphPad Prism Version 5.0d. Gray-filled boxes represent significant upregulation in expression. Asterisks represent p-values (*
Epigenetics Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/kepi20
Dysregulated Class I histone deacetylases are indicators of poor prognosis in multiple myeloma a
b
b
a
abc
Sridurga Mithraprabhu , Anna Kalff , Annie Chow , Tiffany Khong & Andrew Spencer a
Myeloma Research Group; Division of Blood Cancers; Australian Center for Blood Diseases; Alfred Hospital; Monash University; Melbourne, Australia b
Malignant Hematology and Stem Cell Transplantation; Alfred Hospital; Melbourne, Australia
c
Department of Clinical Hematology; Monash University; Clayton, Australia Accepted author version posted online: 06 Dec 2014.Published online: 18 Dec 2014.
Click for updates To cite this article: Sridurga Mithraprabhu, Anna Kalff, Annie Chow, Tiffany Khong & Andrew Spencer (2014) Dysregulated Class I histone deacetylases are indicators of poor prognosis in multiple myeloma, Epigenetics, 9:11, 1511-1520, DOI: 10.4161/15592294.2014.983367 To link to this article: http://dx.doi.org/10.4161/15592294.2014.983367
PLEASE SCROLL DOWN FOR ARTICLE Taylor & Francis makes every effort to ensure the accuracy of all the information (the “Content”) contained in the publications on our platform. However, Taylor & Francis, our agents, and our licensors make no representations or warranties whatsoever as to the accuracy, completeness, or suitability for any purpose of the Content. Any opinions and views expressed in this publication are the opinions and views of the authors, and are not the views of or endorsed by Taylor & Francis. The accuracy of the Content should not be relied upon and should be independently verified with primary sources of information. Taylor and Francis shall not be liable for any losses, actions, claims, proceedings, demands, costs, expenses, damages, and other liabilities whatsoever or howsoever caused arising directly or indirectly in connection with, in relation to or arising out of the use of the Content. This article may be used for research, teaching, and private study purposes. Any substantial or systematic reproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in any form to anyone is expressly forbidden. Terms & Conditions of access and use can be found at http:// www.tandfonline.com/page/terms-and-conditions
RESEARCH PAPER Epigenetics 9:11, 1511--1520; November 2014; © 2014 Taylor & Francis Group, LLC
Dysregulated Class I histone deacetylases are indicators of poor prognosis in multiple myeloma Sridurga Mithraprabhu1, Anna Kalff2, Annie Chow2, Tiffany Khong1, and Andrew Spencer1,2,3,* 1
Myeloma Research Group; Division of Blood Cancers; Australian Center for Blood Diseases; Alfred Hospital; Monash University; Melbourne, Australia; 2Malignant Hematology and Stem Cell Transplantation; Alfred Hospital; Melbourne, Australia; 3Department of Clinical Hematology; Monash University; Clayton, Australia
Keywords: Class I HDAC, HDAC expression, hematological malignancy, histone deacetylases, multiple myeloma, prognosis
Downloaded by [Tulane University] at 22:10 30 January 2015
Abbreviations:
Histone deacetylases (HDAC) control gene expression through their ability to acetylate proteins, thereby influencing a diverse range of cellular functions. Class I HDAC (HDAC1–3 and 8) and HDAC6 are predominantly upregulated in malignancies and their altered expression in some cancers has a significant prognostic implication. The expression and prognostic consequence of dysregulated Class I HDAC and HDAC6, key players in multiple myeloma (MM), are unknown. This study hypothesized that HDAC are dysregulated in MM and patients with high expression have significantly poorer prognostic outcomes. Quantitative PCR for 11 HDAC (Class I, II, and IV) was performed in genetically heterogeneous human myeloma cell lines (HMCL) and primary MM and compared to normal plasma cells (PC). In HMCL, HDAC1–3 and 8 (Class I), and HDAC5 and HDAC10 (Class II) were significantly upregulated compared to normal PC. In primary MM, the median expression level of all of the HDAC, except HDAC1 and HDAC11, were elevated when compared to normal PC. Patients with higher levels of HDAC1–3, HDAC4, HDAC6, and HDAC11 transcripts demonstrated a significantly shorter progression-free survival (PFS). Immunohistochemical staining for HDAC1 and HDAC6 on bone marrow trephines from a uniformly treated cohort of transplant eligible MM patients revealed that HDAC1 protein was detectable in most patients and that higher levels of MM cell HDAC1 protein expression (90 % versus 20 % MM cell positivity) correlated with both shorter PFS (P D 0 .07) and shorter overall survival (P D 0 .003). Conversely, while the majority of patients expressed HDAC6, there was no correlation between HDAC6 levels and patient outcome. Together, these results indicate that overexpression of Class I HDAC, particularly HDAC1, is associated with poor prognosis in MM.
Introduction Multiple myeloma (MM) is an incurable clonal B-cell malignant neoplasm characterized by de novo drug resistance and complex cytogenetic abnormalities that are associated with unique clinical and prognostic implications.1-5 In contrast to the genomic abnormalities, limited information is known about the role of the epigenome in MM pathogenesis and maintenance. Epigenetic modifications, such as DNA methylation and histone acetylation of structurally intact genes have been recognized as critical facets of cancer pathogenesis and maintenance.6 Acetylation is modulated by the dynamic and antagonistic action of 2 classes of enzymes, histone deacetylases (HDAC) and histone acetyltransferases (HAT), wherein HDAC catalyze the removal of acetyl groups and HAT acetylate the N-terminal lysine residues. HDACs are a highly conserved group of enzymes currently consisting of 18 genes grouped into 4 classes, based on their homology to yeast orthologues. Class I (HDAC1–3 and 8), Class IIA (HDAC4, 5, 7, and 9), Class IIB (HDAC6 and 10) and Class IV
(HDAC11) require zinc for catalyzing deacetylase activity, and Class III (Sirtuins 1–7) utilize nicotine adenine dinucleotide (NADC) for their catalytic mechanisms.7,8 HDACs orchestrate a myriad of cellular functions, including proliferation, differentiation, and apoptosis, through the deacetylation of histones and non-histone proteins. Dysregulation of HDAC expression, predominantly overexpression, has been observed in a number of malignancies.9 Class I HDAC expression, in particular, is known to be increased in a number of cancers, including gastric, prostate, colon, breast, renal, and cervical.10-16 Specific to hematological malignancies, dysregulated HDAC expression has been reported in peripheral T-cell lymphomas (PTCL), cutaneous T-cell lymphomas (CTCL), diffuse large B-cell lymphomas (DLBCL), pediatric acute lymphoblastic leukemia (ALL), and myeloproliferative neoplasms.17-20 In all instances, the expression of one or more of Class I HDACs was increased. Prognostic correlates of upregulated HDAC expression are, however, more complex and appear to be highly context-dependent, with the majority of studies
*Correspondence to: Andrew Spencer; Email: [email protected] Submitted: 08/14/2014; Revised: 09/28/2014; Accepted: 10/03/2014 http://dx.doi.org/10.4161/15592294.2014.983367
www.landesbioscience.com
Epigenetics
1511
Downloaded by [Tulane University] at 22:10 30 January 2015
demonstrating a worse prognosis with higher levels of HDAC1 and/or HDAC2 expression.10-14,21 The opposite effect was seen in breast cancer (HDAC1), ALL, and chronic lymphocytic leukemia (CLL; HDAC3), in which overexpression was a favorable prognostic indicator.16,22,23 HDAC6 has also been studied in cancers because of its to ability to orchestrate a variety of cellular processes that are crucial for cancer pathogenesis.24 Overexpression of HDAC6 has been demonstrated in hepatocellular carcinomas,25 CTCL,17 ALL, 22 and breast cancers.26,27 Moreover, in contrast to increased Class I HDAC expression, which for the most part is an indicator of inferior survival, overexpression of HDAC6 has been largely associated with both improved overall (OS) and progression-free survival (PFS), including in studies of CTCL, 17 breast cancer,26,27 lung cancer,28 DLBCL, 20 and CLL.23 Conversely, a small number of studies have demonstrated that increased HDAC6 expression (breast carcinomas and PTCL) is a negative prognostic factor.15,20 In MM, neither the pattern of expression of HDAC nor any potential association with prognosis has been systematically studied. In vitro HDAC6 appears to be a key modulator of MM cell survival, with studies showing the efficacy of HDAC6-selective inhibitors to induce cell death in MM cells.29-31 Similarly, Class I HDAC also play a critical role in MM cell survival, with evidence showing that inhibition of Class I HDAC induces MM cell death and that inhibition of Class I HDAC is superior to HDAC6 inhibition alone in promoting MM cell apoptosis.32,33 Dysregulation of HDAC may also to contribute to MM drug resistance, with overexpression of HDAC1 possibly conferring resistance to bortezomib-induced apoptosis. This resistance is reversed by the simultaneous addition of FK228, a Class I HDAC inhibitor (HDACi).33 Given the role HDACs play in tumor cell survival and drug resistance and the emergence of HDACi as a potentially available class of anti-MM therapeutics, characterizing the patterns of HDAC expression in MM, and any correlation with patient outcome, is of interest.
Results HDAC levels in HMCL are significantly elevated The levels of expression of HDAC1–11 were determined by qRT-PCR in a genetically heterogeneous panel (n D 14 ) of HMCL and compared to the levels of expression in a panel of normal PC (n D 9 ). All the HDAC transcripts were detected in the tested HMCL, with HDAC11 showing the highest expression and the most variability (mean § SEM: 21.29 § 13.66), followed by HDAC1 (5.89 § 1.03), and HDAC5 (4.96 § 0.56) (Fig. 1). The mean values represent the relative expression levels of the particular gene in HMCL compared to normal PC, which have been normalized to one. Moreover, all Class I HDAC isoforms (HDAC1, 2, 3, and 8), HDAC5, and HDAC10 were significantly elevated in HMCL when compared to normal PC (Fig. 1). To determine if HDAC expression showed any correlation with recognized recurring cytogenetic abnormalities, the HMCL
1512
were grouped based on their translocation status and expression levels of HDAC in these groups were compared. Primary IgH translocations in MM are represented predominantly by 4 recurring abnormalities: 4p16 [t(4;14); FGFR3 and MMSET], 11q13 [t(11;14); CYCLIN D1], 6p21 [t(6;14); CYCLIN D3] and 16q23 [t(14;16); C-MAF]. Among the HMCL chosen for this study, 5/14 harbor t(4;14) (OPM-2, NCI-H929, LP-1, KMS18, and JIM-1), 4/14 have t(11;14) (KMS-12BM, KMS-12PE, XG-1, and U266), 3/14 have t(14;16) (OCI-MY1, RPMI-8226, and ANBL-6), and 2/14 have both t(4;14) and t(14;16) (KMS11 and KMS-26).34-38 KMS-11 and KMS-26, which have more than one primary translocation, were not used for analysis. The t (4;14) HMCL had the highest number of HDAC that were overexpressed (8/11; 72.72%) when compared to normal PC, with all of Class I HDAC, HDAC5, HDAC7, HDAC10, and HDAC11 significantly elevated (Table 1). Class I HDAC, HDAC5, and HDAC7 were also significantly elevated in t(11;14) HMCL (6/11; 54.54%), while t(14;16) HMCL showed overexpression of HDAC1, HDAC2, HDAC3, HDAC5, and HDAC10 and downregulation of HDAC6 (6/11; 54.54%). Secondary translocations, which are more complex and associated with tumor progression, are also prevalent in HMCL, most notably involving 8q24 (c-myc). Eleven of the HMCL used in this study have a translocation involving the c-myc locus (the exceptions being U266, ANBL-6, and JIM-1).39,40 The expression of the majority of the HDAC tested (7/11; 63.63%) was also significantly elevated in these HMCL (Table 1). Notably, HDAC1–3 were elevated in all HMCL irrespective of the translocations. These results demonstrate that HDAC are broadly dysregulated in MM with the dysregulation of the Class I HDACs being most prominent, but with no apparent correlation between the pattern of HDAC dysregulation and any particular karyotypic subsets of MM. HDAC are dysregulated in primary MM, with higher expression correlating to significantly shorter survival To determine if HDAC expression is abnormal in primary MM compared to normal plasma cells cohorts of purified MM tumor cells from both newly diagnosed and relapsed MM patients were evaluated. Table 2 illustrates the patient characteristics of the studied MM population. Analyses of gene expression levels of HDAC in primary MM cells (n D 55 ; clinical details available for n D 49 ) and normal PC (n D 9 ) revealed that the median levels of expression of all HDAC, with the exception of HDAC1, HDAC4, and HDAC11, were significantly elevated in MM, compared to normal PC, in which expression levels were normalized to one for that particular gene (Fig. 2). Median HDAC4 expression levels were reduced in comparison to normal PC. HDAC9 had the highest fold change in median expression levels when compared to normal PC (median 3.31) followed by HDAC3 (median 2.23) and HDAC7 (median: 2.20). The potential prognostic significance of dysregulated HDAC expression was then analyzed. The overall survival (OS – from diagnosis to time of death) and progression-free survival (PFS – from diagnosis to time of progression or death) in MM patients with high expression levels ( 75th centile) compared to low levels ( 75th
Epigenetics
Volume 9 Issue 11
Downloaded by [Tulane University] at 22:10 30 January 2015
centile) were derived utilizing Kaplan-Meier survival plots. Of the n D 55 MM samples used for qRT-PCR, 37 had information available to determine OS and PFS. Although the median level of expression of HDAC1 in MM patients was not significantly altered compared to normal PC, those patients with elevated HDAC1 expression had shorter PFS compared to patients with low expression (median PFS; High 586 vs Low 1050; P D 0 .0021; Fig. 3A). Similarly, MM patients with elevated HDAC2, HDAC4, HDAC6, and HDAC11 also showed shorter PFS compared to patients with lower expression levels (Fig. 3B-E) indicating that overexpression of HDAC is a predictor for poor prognosis.
Figure 1. HDAC genes are overexpressed in human myeloma cell lines when compared to normal plasma cells Expression levels of HDAC in 14 human myeloma cell lines (HMCL) were compared to normal plasma cells (normal; n D 9 ). Class I HDAC (HDAC1, HDAC2, HDAC3, and HDAC8) were all significantly overexpressed. Among the class II HDAC, HDAC5 and HDAC10 were significantly overexpressed. Data are presented as mean§SEM. Significant differences were calculated with t-test utilizing GraphPad Prism 5.0d. p-values are indicated.
Increased HDAC1 protein expression correlates with poor overall and progression-free survival The qRT-PCR data from the primary MM cohort demonstrated that overexpression of Class I HDAC genes is significantly correlated with a poorer prognosis. To validate our findings at the protein level, a preliminary analysis of HDAC1–3 and HDAC6 protein expression in BM core biopsies from MM patients was first performed to determine the feasibility of such an approach. HDAC2 and HDAC3 protein expression was ubiquitous, being seen in all MM cells and in a majority of non-MM
cells (data not shown); therefore, it was considered that a correlative analysis of HDAC2 and HDAC3 protein expression would be uninterpretable. In contrast, HDAC1 and HDAC6 expression was more MM cell-restricted, allowing MM cell specific determination to be undertaken. Consequently, validation via analysis of HDAC1 and HDAC6 MM cell protein expression was undertaken utilizing a second independent cohort of 97 uniformly treated transplant-eligible MM patients. Of the 97 tested patients, 79 patients showed assessable HDAC1 nuclear staining of MM cells and, within this cohort, 73.4% (58/79) of patients showed 50 % staining (Table 3). Representative images showing 20% HDAC1 staining and a second specimen with 90%
Table 1. HDAC1-2 (Class I HDAC) are overexpressed in all HMCL Primary translocations Gene HDAC1 HDAC2 HDAC3 HDAC8 HDAC5 HDAC7 HDAC6 HDAC10 HDAC11
Secondary translocations
Normal
t(4;14) (FGFR3 and MMSET)
t(11;14) (CYCLIN D1)
t(14;16) (C-MAF)
8q24 (c-MYC)
1.15§0.25 1.18§0.26 1.24§0.29 1.21§0.24 1.36§0.32 1.30§0.33 1.21§0.81 1.12§0.17 2.59§1.06
5.75§0.80**** 4.83§0.48**** 3.44§0.76** 2.03§0.12** 5.41§0.70**** 2.57§0.31** 1.57§0.26 2.76§0.26**** 54.10§26.85*
7.73§2.04**** 5.13§0.88**** 4.00§0.71*** 2.29§0.36* 6.23§1.24*** 2.53§0.20* 2.70§0.93 1.47§0.34 7.85§2.73
3.17§0.41*** 2.21§0.31** 2.65§0.23** 1.66§0.17 3.15§0.54** 1.61§0.17 0.69§0.08* 2.25§0.36* 1.77§0/36
6.67§0.95**** 4.33§0.39**** 3.68§0.44**** 2.29§0.15*** 5.32§0.57**** 2.68§0.31** 1.82§0.37 2.53§0.22** 9.93§3.37
HMCLs were grouped based on their translocation status [t(4;14), t(11;14), t(14;16) and translocations involving c-myc locus – 8q24] and expression levels of HDAC were compared to those in normal plasma cells. HMCL with t(4;14) had significantly higher levels of HDAC (8/11) followed by translocations involving c-myc locus (7/11). HMCLs with t(11;14) and t(14;16) had 6/11 HDACs significantly dysregulated. Significant differences were observed using t-test and GraphPad Prism Version 5.0d. Gray-filled boxes represent significant upregulation in expression. Asterisks represent p-values (*
