Letters to the Editor
455 to NEK. We also acknowledge Tolero for providing us with the Axl inhibitor SGI-7079 and excellent secretarial help from Ms Tammy Hughes.
J Boysen1,3, S Sinha1,3, T Price-Troska1, SL Warner2, DJ Bearss2, D Viswanatha1, TD Shanafelt1, NE Kay1 and AK Ghosh1 1 Division of Hematology, Mayo Clinic, Rochester, MN, USA and 2 Tolero Pharmaceuticals Inc., Salt Lake City, UT, USA E-mail: [email protected] 3 These authors contributed equally to this work. REFERENCES 1 Ghosh AK, Secreto C, Boysen J, Sassoon T, Shanafelt TD, Mukhopadhyay D et al. The novel receptor tyrosine kinase Axl is constitutively active in B-cell chronic lymphocytic leukemia and acts as a docking site of nonreceptor kinases: implications for therapy. Blood 2011; 117: 1928–1937. 2 Hermeking H. The miR-34 family in cancer and apoptosis. Cell Death Differ 2010; 17: 193–199. 3 Ghosh AK, Boysen J, Price-troska T, Secreto C, Zent CS, Kay N. Axl receptor tyrosine kinase signaling pathway and the p53 tumor suppressor protein exist in a novel regulatory loop in B-cell chronic lymphocytic leukemia cells. ASH Annu Meet Abstr 2011; 118: 799. 4 Mudduluru G, Ceppi P, Kumarswamy R, Scagliotti GV, Papotti M, Allgayer H. Regulation of Axl receptor tyrosine kinase expression by miR-34a and miR-199a/b in solid cancer. Oncogene 2011; 30: 2888–2899. 5 Mackiewicz M, Huppi K, Pitt JJ, Dorsey TH, Ambs S, Caplen NJ. Identification of the receptor tyrosine kinase AXL in breast cancer as a target for the human miR-34a microRNA. Breast Cancer Res Treat 2011; 130: 663–679.
6 Zenz T, Mohr J, Eldering E, Kater AP, Buhler A, Kienle D et al. MiR-34a as part of the resistance network in chronic lymphocytic leukemia. Blood 2009; 113: 3801–3808. 7 Zenz T, Habe S, Denzel T, Mohr J, Winkler D, Buhler A et al. Detailed analysis of p53 pathway defects in fludarabine-refractory chronic lymphocytic leukemia (CLL): dissecting the contribution of 17p deletion, TP53 mutation, p53-p21 dysfunction, and miR34a in a prospective clinical trial. Blood 2009; 114: 2589–2597. 8 Dijkstra MK, van Lom K, Tielemans D, Elstrodt F, Langerak AW, van ’t Veer MB et al. 17P13/TP53 deletion in B-CLL patients is associated with microRNA-34a downregulation. Leukemia 2009; 23: 625–627. 9 Hirao A, Cheung A, Duncan G, Girard PM, Elia AJ, Wakeham A et al. Chk2 is a tumor suppressor that regulates apoptosis in both an ataxia telangiectasia mutated (ATM)-dependent and an ATM-independent manner. Mol Cell Biol 2002; 22: 6521–6532. 10 Tibbetts RS, Brumbaugh KM, Williams JM, Sarkaria JN, Cliby WA, Shieh SY et al. A role for ATR in the DNA damage-induced phosphorylation of p53. Genes Dev 1999; 13: 152–157. 11 Chim CS, Wong KY, Qi Y, Loong F, Lam WL, Wong LG et al. Epigenetic inactivation of the miR-34a in hematological malignancies. Carcinogenesis 2010; 31: 745–750. 12 Lodygin D, Tarasov V, Epanchintsev A, Berking C, Knyazeva T, Korner H et al. Inactivation of miR-34a by aberrant CpG methylation in multiple types of cancer. Cell Cycle 2008; 7: 2591–2600. 13 Chang TC, Wentzel EA, Kent OA, Ramachandran K, Mullendore M, Lee KH et al. Transactivation of miR-34a by p53 broadly influences gene expression and promotes apoptosis. Mol Cell 2007; 26: 745–752. 14 Kay NE, O’Brien SM, Pettitt AR, Stilgenbauer S. The role of prognostic factors in assessing ‘high-risk’ subgroups of patients with chronic lymphocytic leukemia. Leukemia 2007; 21: 1885–1891. 15 Mudduluru G, Allgayer H. The human receptor tyrosine kinase Axl gene—promoter characterization and regulation of constitutive expression by Sp1, Sp3 and CpG methylation. Biosci Rep 2008; 28: 161–176.
Supplementary Information accompanies this paper on the Leukemia website (http://www.nature.com/leu)
Long-term response to lenalidomide in patients with newly diagnosed multiple myeloma Leukemia (2014) 28, 455–457; doi:10.1038/leu.2013.300
Lenalidomide is an immunomodulatory (IMid) drug that is commonly used to treat newly diagnosed and relapsed multiple myeloma (MM).1 Clinical observations in patients with newly diagnosed MM suggest that some patients are able to attain longterm remissions after treatment with immunomodulatory drugs, some of which may be sustained for several years.2 In this study, our goal was to identify predictive factors among those patients who were induced with and continued on lenalidomide for X3 years (long-term responders (LTRs)). Between 2003 and 2011, 305 patients with newly diagnosed active MM, who received initial treatment with lenalidomide and dexamethasone, were seen at the Mayo Clinic, Rochester. Complete clinical, laboratory and treatment data were available for 284 patients and were extracted after retrospective review of their medical records. The Mayo Foundation Institutional Review Board approved the study, and all patients consented to have their medical records reviewed according to the institutional review board practices. One hundred and nine patients were excluded leaving 175 patients for the current study. Reasons for exclusion included early autologous stem cell transplant in 103 and less than 3 years of follow-up in 6. Patients were treated with lenalidomide 25 mg daily for 21 days of a 28-day cycle. Initial patients were treated with high-dose dexamethasone (40 mg on
days 1–4, 9–12, 17–20 of a 28-day cycle), which was changed to 40 mg weekly once the results of the E4A03 clinical trial were reported.3 All patients had disease assessment after each cycle of treatment, and the International Myeloma Working Group (IMWG) uniform response criteria were used to assess disease response. Outside of a clinical trial setting, a bone marrow was not always performed for confirmation of a complete response (CR). Overall survival (OS) and progression-free survival (PFS) were estimated by the method of Kaplan–Meier. The Pearson w2-test and the Kruskal–Wallis tests were used to ascertain differences between nominal and continuous variables, respectively. P-values less than 0.05 were considered significant. All statistics were done using JMP software (SAS, Carey, NC, USA). The baseline characteristics and long-term outcomes for the whole cohort have already been described.4 Herein we focus on outcomes of LTRs (Table 1). Of the 175 patients in this study, a total of 33 (19%) received lenalidomide for more than 3 years. Median PFS from diagnosis for LTRs was 102 months and median OS was not reached. Overall, 94% (31/33) patients were alive after a median follow-up of 56 months. Of the LTRs, 19 patients remain on the drug after a median follow-up of 52 months. Eight discontinued the drug because of progressive disease after a median follow-up of 82 months and four because of prolonged disease stability (SD) after a median follow-up of 71 months and were followed off treatment. Two patients discontinued for reasons other than progression or SD and were followed off treatment; one patient after 102 months because of
Accepted article preview online 22 Ocober 2013; advance online publication, 12 November 2013
& 2014 Macmillan Publishers Limited
Leukemia (2014) 404 – 463
Letters to the Editor
456 Table 1.
Baseline characteristics of patients according to duration of lenalidomide
Total, N ¼ 175 Median (range) Age Male sex, N (%) Diagnosed in or after 2008 Performance status Serum M-spike (g/dl) Involved dFLC (mg/dl) CRP (mg/l) LDH (U/l) Serum albumin (g/dl) B2 microglobulin (mg/ml) ISS
Lenalidomide o3 years,a n ¼ 142 66 90 69 1 2.8 49 0.3 159 3.5 3.7 2
(32–93) (63%) (49%) (0–4) (0.01–6.9) (0–2368) (0.02–9.2) (88–331) (2.1–4.8) (1.3–25.6) (1–3)
FISH risk Standard Intermediate High
52 (44%) 44 (38%) 21 (18%)
Plasma cell labeling index (%) Dexamethasone schedule 12 days/cycle (high dose) 4 days/cycle (low dose) Duration of Lenalidomide Best response Greater VGPR PR SD PD Time to best response, months Follow up, months Overall survival, monthsb
Lenalidomide X3 years, n ¼ 33 65 21 18 1 3.2 27 0.3 154 3.6 3.3 2
(35–87) (64%) (55%) (0–2) (0.01–7.6) (0.08–1060) (0.08–11.3) (102–268) (2.6–4.5) (2–11) (1–3)
P-value 0.4 0.98 0.54 0.26 0.83 0.11 0.13 0.52 0.48 0.21 0.07
18 (64%) 9 (32%) 1 (4%)
0.03 0.15 0.04
0.8 (0–9.4)
1 (0–11)
0.31
24 (17%) 118 (83%) 7 (0.1–35)
9 (27%) 24 (73%) 53 (39–103)
0.22 0.22 o0.001
21 (64%) 10 (30%) 2 (6%) 0 4 (1–16) 57 (40–111) Not reached
o0.001 0.43 0.02 0.02 o0.001 o0.001 o0.001
31 54 34 12 6 43
(22%) (38%) (24%) (9%) (2–40) (0.8–107) 52
Abbreviations: CRP, C reactive protein; dFLC, difference between involved and uninvolved free light chains; FISH: fluorescent in situ hybridization; LDH, lactate dehydrogenase; PD, progressive disease; PR: partial response; SD, stable disease; VGPR, very good partial response. aIncludes patients who had follow-up for 43 years but used Lenalidomide for o3 years or patients who died earlier than 3 years. Patients lost to follow-up earlier than 3 years were not included. b Calculated with the method of Kaplan–Meier.
an intracranial hemorrhage not related to lenalidomide and the other after 62 months because of refractory diarrhea that was multifactorial and improved upon stopping the drug. When compared with patients receiving lenalidomide for less than 3 years, LTRs were more likely to have standard risk5 disease (64% versus 44%, P ¼ 0.03) and less likely to have high-risk disease (18% versus 4%, P ¼ 0.04). Depth of response differed between the two groups, with 64% (21/33) of the LTR group in very good partial response (VGPR) or better (11 patients with confirmed CR) versus 22% (31/142) in the remainder, Po0.0001. Furthermore, LTRs had a longer median time to achieving best response (6 versus 4 months, Po0.001). We next considered the 12 patients who received lenalidomide for more than 5 years and excluded 55 patients who had less than 5 years of follow-up for these analyses. In this group, 83% (10/12) achieved VGPR or better (8 with confirmed CR) versus 22% (23/108) in the remainder, Po0.0001. Median time to achieving best response was also longer (9 versus 4 months, Po0.0001) than those 108 patients who remained on lenalidomide for less than 5 years. In this report, we demonstrated that approximately one out of five patients with newly diagnosed MM can achieve responses lasting more than 3 years while on treatment with lenalidomide. Not surprisingly6 patients with standard risk cytogenetics and achieving greater VGPR were more likely to be LTRs. Long-term use of lenalidomide was also safe, with only one patient stopping for toxicity. Furthermore, LTRs were more likely to be slow responders. This is consistent with previous observations that Leukemia (2014) 404 – 463
suggest that chemosensitive plasma cell clones tend to behave more aggressively in that, although they respond faster to treatment, they are also more likely to relapse earlier.7,8 We can only hypothesize about the potential mechanisms of these durable remissions, but the immunomodulatory actions of lenalidomide are likely implicated.
CONFLICT OF INTEREST Shaji K Kumar is a consultant for Celgene, Millennium Pharmaceuticals, Onyx. Morie Gertz and Martha Q Lacy have received honoraria from Celgene.
ACKNOWLEDGEMENTS This work was supported in part by the JABBS Foundation, the Predolin Foundation and the Robert A Kyle Hematologic Malignancies Fund. Research funding was provided to Angela Dispenzieri by Celgene, Janssen Pharmaceuticals and Millennium Pharmaceuticals. Shaji K Kumar also received research funding from Celgene, Millennium Pharmaceuticals, Onyx.
AUTHOR CONTRIBUTIONS Conception and design: Angela Dispenzieri; provision of study materials of patients: all authors; collection and assembly of data: all authors; data analysis and interpretation: Taxiarchis V Kourelis, Angela Dispenzieri, Shaji Kumar; writing of manuscript and final approval of manuscript: all authors.
& 2014 Macmillan Publishers Limited
Letters to the Editor
457 TV Kourelis, SK Kumar, G Srivastava, MA Gertz, MQ Lacy, FK Buadi, RA Kyle and A Dispenzieri Division of Hematology, Department of Medicine, Mayo Clinic, Rochester, MN, USA E-mail: [email protected] REFERENCES 1 Dimopoulos MA, Kastritis E, Rajkumar SV. Treatment of plasma cell dyscrasias with lenalidomide. Leukemia 2008; 22: 1343–1353. 2 Richardson PG, Laubach JP, Schlossman RL, Ghobrial IM, Redman KC, McKenney M et al. The potential benefits of participating in early-phase clinical trials in multiple myeloma: long-term remission in a patient with relapsed multiple myeloma treated with 90 cycles of lenalidomide and bortezomib. Eur J Haematol 2012; 88: 446–449. 3 Rajkumar SV, Jacobus S, Callander NS, Fonseca R, Vesole DH, Williams ME et al. Lenalidomide plus high-dose dexamethasone versus lenalidomide plus low-dose dexamethasone as initial therapy for newly diagnosed multiple myeloma: an open-label randomised controlled trial. Lancet Oncol 2010; 11: 29–37.
4 Srivastava G, Rana V, Lacy MQ, Buadi FK, Hayman SR, Dispenzieri A et al. Long-term outcome with lenalidomide and dexamethasone therapy for newly diagnosed multiple myeloma. Leukemia 2013; 27: 2062–2066. 5 Mikhael JR, Dingli D, Roy V, Reeder CB, Buadi FK, Hayman SR et al. Management of newly diagnosed symptomatic multiple myeloma: updated Mayo Stratification of Myeloma and Risk-Adapted Therapy (mSMART) consensus guidelines 2013. Mayo Clinic proc 2013; 88: 360–376. 6 Dingli D, Pacheco JM, Nowakowski GS, Kumar SK, Dispenzieri A, Hayman SR et al. Relationship between depth of response and outcome in multiple myeloma. J Clin Oncol 2007; 25: 4933–4937. 7 van Rhee F, Bolejack V, Hollmig K, Pineda-Roman M, Anaissie E, Epstein J et al. High serum-free light chain levels and their rapid reduction in response to therapy define an aggressive multiple myeloma subtype with poor prognosis. Blood 2007; 110: 827–832. 8 Boccadoro M, Marmont F, Tribalto M, Fossati G, Redoglia V, Battaglio S et al. Early responder myeloma: kinetic studies identify a patient subgroup characterized by very poor prognosis. J Clin Oncol 1989; 7: 119–125.
Induction of differential apoptotic pathways in multiple myeloma cells by class-selective histone deacetylase inhibitors Leukemia (2014) 28, 457–460; doi:10.1038/leu.2013.301
Histone deacetylase (HDAC) 6, a class-IIb HDAC, is the only HDAC with two functional deacetylase domains and a zinc-finger motif. HDAC6 was initially described as tubulin deacetylase; however, it also deacetylates other non-histone proteins, including heat-shock protein (Hsp90). Importantly, HDAC6 has an essential role in the aggresomal protein degradation pathway, an alternative system to the proteasome for degradation of polyubiquitinated misfolded/unfolded proteins. Specifically, HDAC6 binds to both polyubiquitinated proteins and dynein motors, thereby acting to recruit protein cargo to dynein motors for transport to aggresomes for lysosomal degradation.1 We have previously shown that inhibition of both proteasomal and aggresomal protein degradation using bortezomib (BTZ) to inhibit the proteasome and tubacin to inhibit HDAC6, respectively, induces accumulation of polyubiquitinated proteins and significant multiple myeloma (MM) cell stress, followed by apoptosis.2 More recently, a hydroxamic acid HDAC6-selective inhibitor ACY-1215 combined with BTZ shows significant anti-MM activities in preclinical studies,3 which have been rapidly translated to phase I/II clinical studies to treat relapsed/refractory MM.4 Previous studies have also shown that the non-selective HDAC inhibitor vorinostat in combination with BTZ also triggers synergistic cytotoxicity in MM,5 which has provided the framework for combination clinical trials.6 Carfilzomib (CFZ) is an epoxyketone proteasome inhibitor, which recently received accelerated Food and Drug Administration approval to treat relapsed refractory MM.7,8 In this study, we asked whether HDAC6 inhibitors enhance anti-MM toxicity induced by CFZ. We also delineated differential molecular mechanisms that trigger synergistic MM toxicity triggered by CFZ in combination with either HDAC6-selective or class-I HDAC-selective inhibitors ACY-1215 and MS275 (entinostat), respectively.9 We first compared the combination effect of the HDAC6-selective inhibitor ACY-1215 with either BTZ or CFZ in MM cell lines. Synergistic cytotoxicity induced by CFZ with ACY-1215 was greater than that by BTZ with ACY-1215 in both RPMI8226 (Figure 1a, upper panel) and MM.1S cells (Figure 1a, lower panel). To further confirm the role of HDAC6 inhibition, we carried out similar combination cytotoxicity experiments using the chemically
unrelated HDAC6-selective inhibitor tubastatin-A. Consistent with the ACY-1215 results, tubastatin-A synergistically enhanced both BTZ- and CFZ-induced cytotoxicity. Interestingly, synergistic cytotoxicity, on the basis of combination index (CI) o1, was more significant in RPMI8226 cells than in MM.1S cells (Supplementary Figure 1). Although BTZ has a remarkable clinical activity in MM, acquired resistance limits its long-term utility. We, therefore, next examined whether HDAC6 inhibition can overcome BTZ resistance in vitro using the BTZ-resistant ANBL (ANBL-R) human MM cell line. Importantly, the synergistic cytotoxicity of ACY-1215 with BTZ was observed even in ANBL-R cells. Similar to Figure 1a, this synergistic cytotoxicity was even more significant with CFZ compared with BTZ (Figure 1b). These results suggest that CFZ may have a greater clinical activity in MM than in BTZ when combined with HDAC6 inhibitors. We and others have shown that non-selective HDAC inhibitors in combination with BTZ trigger synergistic cytotoxicity in MM.10,11 These non-selective HDAC inhibitors block not only HDAC6 but also the class-I HDAC activity,9 and whether synergistic cytotoxicity induced by non-selective HDAC inhibitors with BTZ is due solely to HDAC6 blockade remains unclear. Indeed, we have recently shown that both HDAC3 knockdown and a smallmolecule HDAC3-selective inhibitor BG45 trigger synergistic MM cytotoxicity in combination with BTZ.12 To address this question, we first examined the cytotoxicity of class-I HDAC-selective inhibitor MS275 in combination with CFZ. MS275 with CFZ induced synergistic cytotoxicity in RPMI8226 cells and additive cytotoxicity in MM.1S cells (Supplementary Figure 2). Consistent with our studies, MS275 with BTZ also shows significant antitumor activities in other cancer cell types.13 We next determined whether different molecular mechanisms mediate the synergistic MM cell cytotoxicity of HDAC6 versus class-I HDAC inhibition in combination with CFZ. We first defined the doses of either ACY-1215 (2 mM) or MS275 (2 mM) with CFZ (10 nM) that induced equivalent cytotoxicity (35%) (Supplementary Figure 3) and used these culture conditions for subsequent experiments. We have shown that ACY-1215 with BTZ triggers the accumulation of polyubiquitinated proteins associated with cell stress.3 Although not all polyubiquitinated proteins are degraded by the proteasome, recent studies have shown that proteins specifically linked with lysine (K)48 polyubiquitin are degraded by proteasomes.14 Our studies showed that CFZ induced
Accepted article preview online 22 October 2013; advance online publication, 12 November 2013
& 2014 Macmillan Publishers Limited
Leukemia (2014) 404 – 463
455 to NEK. We also acknowledge Tolero for providing us with the Axl inhibitor SGI-7079 and excellent secretarial help from Ms Tammy Hughes.
J Boysen1,3, S Sinha1,3, T Price-Troska1, SL Warner2, DJ Bearss2, D Viswanatha1, TD Shanafelt1, NE Kay1 and AK Ghosh1 1 Division of Hematology, Mayo Clinic, Rochester, MN, USA and 2 Tolero Pharmaceuticals Inc., Salt Lake City, UT, USA E-mail: [email protected] 3 These authors contributed equally to this work. REFERENCES 1 Ghosh AK, Secreto C, Boysen J, Sassoon T, Shanafelt TD, Mukhopadhyay D et al. The novel receptor tyrosine kinase Axl is constitutively active in B-cell chronic lymphocytic leukemia and acts as a docking site of nonreceptor kinases: implications for therapy. Blood 2011; 117: 1928–1937. 2 Hermeking H. The miR-34 family in cancer and apoptosis. Cell Death Differ 2010; 17: 193–199. 3 Ghosh AK, Boysen J, Price-troska T, Secreto C, Zent CS, Kay N. Axl receptor tyrosine kinase signaling pathway and the p53 tumor suppressor protein exist in a novel regulatory loop in B-cell chronic lymphocytic leukemia cells. ASH Annu Meet Abstr 2011; 118: 799. 4 Mudduluru G, Ceppi P, Kumarswamy R, Scagliotti GV, Papotti M, Allgayer H. Regulation of Axl receptor tyrosine kinase expression by miR-34a and miR-199a/b in solid cancer. Oncogene 2011; 30: 2888–2899. 5 Mackiewicz M, Huppi K, Pitt JJ, Dorsey TH, Ambs S, Caplen NJ. Identification of the receptor tyrosine kinase AXL in breast cancer as a target for the human miR-34a microRNA. Breast Cancer Res Treat 2011; 130: 663–679.
6 Zenz T, Mohr J, Eldering E, Kater AP, Buhler A, Kienle D et al. MiR-34a as part of the resistance network in chronic lymphocytic leukemia. Blood 2009; 113: 3801–3808. 7 Zenz T, Habe S, Denzel T, Mohr J, Winkler D, Buhler A et al. Detailed analysis of p53 pathway defects in fludarabine-refractory chronic lymphocytic leukemia (CLL): dissecting the contribution of 17p deletion, TP53 mutation, p53-p21 dysfunction, and miR34a in a prospective clinical trial. Blood 2009; 114: 2589–2597. 8 Dijkstra MK, van Lom K, Tielemans D, Elstrodt F, Langerak AW, van ’t Veer MB et al. 17P13/TP53 deletion in B-CLL patients is associated with microRNA-34a downregulation. Leukemia 2009; 23: 625–627. 9 Hirao A, Cheung A, Duncan G, Girard PM, Elia AJ, Wakeham A et al. Chk2 is a tumor suppressor that regulates apoptosis in both an ataxia telangiectasia mutated (ATM)-dependent and an ATM-independent manner. Mol Cell Biol 2002; 22: 6521–6532. 10 Tibbetts RS, Brumbaugh KM, Williams JM, Sarkaria JN, Cliby WA, Shieh SY et al. A role for ATR in the DNA damage-induced phosphorylation of p53. Genes Dev 1999; 13: 152–157. 11 Chim CS, Wong KY, Qi Y, Loong F, Lam WL, Wong LG et al. Epigenetic inactivation of the miR-34a in hematological malignancies. Carcinogenesis 2010; 31: 745–750. 12 Lodygin D, Tarasov V, Epanchintsev A, Berking C, Knyazeva T, Korner H et al. Inactivation of miR-34a by aberrant CpG methylation in multiple types of cancer. Cell Cycle 2008; 7: 2591–2600. 13 Chang TC, Wentzel EA, Kent OA, Ramachandran K, Mullendore M, Lee KH et al. Transactivation of miR-34a by p53 broadly influences gene expression and promotes apoptosis. Mol Cell 2007; 26: 745–752. 14 Kay NE, O’Brien SM, Pettitt AR, Stilgenbauer S. The role of prognostic factors in assessing ‘high-risk’ subgroups of patients with chronic lymphocytic leukemia. Leukemia 2007; 21: 1885–1891. 15 Mudduluru G, Allgayer H. The human receptor tyrosine kinase Axl gene—promoter characterization and regulation of constitutive expression by Sp1, Sp3 and CpG methylation. Biosci Rep 2008; 28: 161–176.
Supplementary Information accompanies this paper on the Leukemia website (http://www.nature.com/leu)
Long-term response to lenalidomide in patients with newly diagnosed multiple myeloma Leukemia (2014) 28, 455–457; doi:10.1038/leu.2013.300
Lenalidomide is an immunomodulatory (IMid) drug that is commonly used to treat newly diagnosed and relapsed multiple myeloma (MM).1 Clinical observations in patients with newly diagnosed MM suggest that some patients are able to attain longterm remissions after treatment with immunomodulatory drugs, some of which may be sustained for several years.2 In this study, our goal was to identify predictive factors among those patients who were induced with and continued on lenalidomide for X3 years (long-term responders (LTRs)). Between 2003 and 2011, 305 patients with newly diagnosed active MM, who received initial treatment with lenalidomide and dexamethasone, were seen at the Mayo Clinic, Rochester. Complete clinical, laboratory and treatment data were available for 284 patients and were extracted after retrospective review of their medical records. The Mayo Foundation Institutional Review Board approved the study, and all patients consented to have their medical records reviewed according to the institutional review board practices. One hundred and nine patients were excluded leaving 175 patients for the current study. Reasons for exclusion included early autologous stem cell transplant in 103 and less than 3 years of follow-up in 6. Patients were treated with lenalidomide 25 mg daily for 21 days of a 28-day cycle. Initial patients were treated with high-dose dexamethasone (40 mg on
days 1–4, 9–12, 17–20 of a 28-day cycle), which was changed to 40 mg weekly once the results of the E4A03 clinical trial were reported.3 All patients had disease assessment after each cycle of treatment, and the International Myeloma Working Group (IMWG) uniform response criteria were used to assess disease response. Outside of a clinical trial setting, a bone marrow was not always performed for confirmation of a complete response (CR). Overall survival (OS) and progression-free survival (PFS) were estimated by the method of Kaplan–Meier. The Pearson w2-test and the Kruskal–Wallis tests were used to ascertain differences between nominal and continuous variables, respectively. P-values less than 0.05 were considered significant. All statistics were done using JMP software (SAS, Carey, NC, USA). The baseline characteristics and long-term outcomes for the whole cohort have already been described.4 Herein we focus on outcomes of LTRs (Table 1). Of the 175 patients in this study, a total of 33 (19%) received lenalidomide for more than 3 years. Median PFS from diagnosis for LTRs was 102 months and median OS was not reached. Overall, 94% (31/33) patients were alive after a median follow-up of 56 months. Of the LTRs, 19 patients remain on the drug after a median follow-up of 52 months. Eight discontinued the drug because of progressive disease after a median follow-up of 82 months and four because of prolonged disease stability (SD) after a median follow-up of 71 months and were followed off treatment. Two patients discontinued for reasons other than progression or SD and were followed off treatment; one patient after 102 months because of
Accepted article preview online 22 Ocober 2013; advance online publication, 12 November 2013
& 2014 Macmillan Publishers Limited
Leukemia (2014) 404 – 463
Letters to the Editor
456 Table 1.
Baseline characteristics of patients according to duration of lenalidomide
Total, N ¼ 175 Median (range) Age Male sex, N (%) Diagnosed in or after 2008 Performance status Serum M-spike (g/dl) Involved dFLC (mg/dl) CRP (mg/l) LDH (U/l) Serum albumin (g/dl) B2 microglobulin (mg/ml) ISS
Lenalidomide o3 years,a n ¼ 142 66 90 69 1 2.8 49 0.3 159 3.5 3.7 2
(32–93) (63%) (49%) (0–4) (0.01–6.9) (0–2368) (0.02–9.2) (88–331) (2.1–4.8) (1.3–25.6) (1–3)
FISH risk Standard Intermediate High
52 (44%) 44 (38%) 21 (18%)
Plasma cell labeling index (%) Dexamethasone schedule 12 days/cycle (high dose) 4 days/cycle (low dose) Duration of Lenalidomide Best response Greater VGPR PR SD PD Time to best response, months Follow up, months Overall survival, monthsb
Lenalidomide X3 years, n ¼ 33 65 21 18 1 3.2 27 0.3 154 3.6 3.3 2
(35–87) (64%) (55%) (0–2) (0.01–7.6) (0.08–1060) (0.08–11.3) (102–268) (2.6–4.5) (2–11) (1–3)
P-value 0.4 0.98 0.54 0.26 0.83 0.11 0.13 0.52 0.48 0.21 0.07
18 (64%) 9 (32%) 1 (4%)
0.03 0.15 0.04
0.8 (0–9.4)
1 (0–11)
0.31
24 (17%) 118 (83%) 7 (0.1–35)
9 (27%) 24 (73%) 53 (39–103)
0.22 0.22 o0.001
21 (64%) 10 (30%) 2 (6%) 0 4 (1–16) 57 (40–111) Not reached
o0.001 0.43 0.02 0.02 o0.001 o0.001 o0.001
31 54 34 12 6 43
(22%) (38%) (24%) (9%) (2–40) (0.8–107) 52
Abbreviations: CRP, C reactive protein; dFLC, difference between involved and uninvolved free light chains; FISH: fluorescent in situ hybridization; LDH, lactate dehydrogenase; PD, progressive disease; PR: partial response; SD, stable disease; VGPR, very good partial response. aIncludes patients who had follow-up for 43 years but used Lenalidomide for o3 years or patients who died earlier than 3 years. Patients lost to follow-up earlier than 3 years were not included. b Calculated with the method of Kaplan–Meier.
an intracranial hemorrhage not related to lenalidomide and the other after 62 months because of refractory diarrhea that was multifactorial and improved upon stopping the drug. When compared with patients receiving lenalidomide for less than 3 years, LTRs were more likely to have standard risk5 disease (64% versus 44%, P ¼ 0.03) and less likely to have high-risk disease (18% versus 4%, P ¼ 0.04). Depth of response differed between the two groups, with 64% (21/33) of the LTR group in very good partial response (VGPR) or better (11 patients with confirmed CR) versus 22% (31/142) in the remainder, Po0.0001. Furthermore, LTRs had a longer median time to achieving best response (6 versus 4 months, Po0.001). We next considered the 12 patients who received lenalidomide for more than 5 years and excluded 55 patients who had less than 5 years of follow-up for these analyses. In this group, 83% (10/12) achieved VGPR or better (8 with confirmed CR) versus 22% (23/108) in the remainder, Po0.0001. Median time to achieving best response was also longer (9 versus 4 months, Po0.0001) than those 108 patients who remained on lenalidomide for less than 5 years. In this report, we demonstrated that approximately one out of five patients with newly diagnosed MM can achieve responses lasting more than 3 years while on treatment with lenalidomide. Not surprisingly6 patients with standard risk cytogenetics and achieving greater VGPR were more likely to be LTRs. Long-term use of lenalidomide was also safe, with only one patient stopping for toxicity. Furthermore, LTRs were more likely to be slow responders. This is consistent with previous observations that Leukemia (2014) 404 – 463
suggest that chemosensitive plasma cell clones tend to behave more aggressively in that, although they respond faster to treatment, they are also more likely to relapse earlier.7,8 We can only hypothesize about the potential mechanisms of these durable remissions, but the immunomodulatory actions of lenalidomide are likely implicated.
CONFLICT OF INTEREST Shaji K Kumar is a consultant for Celgene, Millennium Pharmaceuticals, Onyx. Morie Gertz and Martha Q Lacy have received honoraria from Celgene.
ACKNOWLEDGEMENTS This work was supported in part by the JABBS Foundation, the Predolin Foundation and the Robert A Kyle Hematologic Malignancies Fund. Research funding was provided to Angela Dispenzieri by Celgene, Janssen Pharmaceuticals and Millennium Pharmaceuticals. Shaji K Kumar also received research funding from Celgene, Millennium Pharmaceuticals, Onyx.
AUTHOR CONTRIBUTIONS Conception and design: Angela Dispenzieri; provision of study materials of patients: all authors; collection and assembly of data: all authors; data analysis and interpretation: Taxiarchis V Kourelis, Angela Dispenzieri, Shaji Kumar; writing of manuscript and final approval of manuscript: all authors.
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Letters to the Editor
457 TV Kourelis, SK Kumar, G Srivastava, MA Gertz, MQ Lacy, FK Buadi, RA Kyle and A Dispenzieri Division of Hematology, Department of Medicine, Mayo Clinic, Rochester, MN, USA E-mail: [email protected] REFERENCES 1 Dimopoulos MA, Kastritis E, Rajkumar SV. Treatment of plasma cell dyscrasias with lenalidomide. Leukemia 2008; 22: 1343–1353. 2 Richardson PG, Laubach JP, Schlossman RL, Ghobrial IM, Redman KC, McKenney M et al. The potential benefits of participating in early-phase clinical trials in multiple myeloma: long-term remission in a patient with relapsed multiple myeloma treated with 90 cycles of lenalidomide and bortezomib. Eur J Haematol 2012; 88: 446–449. 3 Rajkumar SV, Jacobus S, Callander NS, Fonseca R, Vesole DH, Williams ME et al. Lenalidomide plus high-dose dexamethasone versus lenalidomide plus low-dose dexamethasone as initial therapy for newly diagnosed multiple myeloma: an open-label randomised controlled trial. Lancet Oncol 2010; 11: 29–37.
4 Srivastava G, Rana V, Lacy MQ, Buadi FK, Hayman SR, Dispenzieri A et al. Long-term outcome with lenalidomide and dexamethasone therapy for newly diagnosed multiple myeloma. Leukemia 2013; 27: 2062–2066. 5 Mikhael JR, Dingli D, Roy V, Reeder CB, Buadi FK, Hayman SR et al. Management of newly diagnosed symptomatic multiple myeloma: updated Mayo Stratification of Myeloma and Risk-Adapted Therapy (mSMART) consensus guidelines 2013. Mayo Clinic proc 2013; 88: 360–376. 6 Dingli D, Pacheco JM, Nowakowski GS, Kumar SK, Dispenzieri A, Hayman SR et al. Relationship between depth of response and outcome in multiple myeloma. J Clin Oncol 2007; 25: 4933–4937. 7 van Rhee F, Bolejack V, Hollmig K, Pineda-Roman M, Anaissie E, Epstein J et al. High serum-free light chain levels and their rapid reduction in response to therapy define an aggressive multiple myeloma subtype with poor prognosis. Blood 2007; 110: 827–832. 8 Boccadoro M, Marmont F, Tribalto M, Fossati G, Redoglia V, Battaglio S et al. Early responder myeloma: kinetic studies identify a patient subgroup characterized by very poor prognosis. J Clin Oncol 1989; 7: 119–125.
Induction of differential apoptotic pathways in multiple myeloma cells by class-selective histone deacetylase inhibitors Leukemia (2014) 28, 457–460; doi:10.1038/leu.2013.301
Histone deacetylase (HDAC) 6, a class-IIb HDAC, is the only HDAC with two functional deacetylase domains and a zinc-finger motif. HDAC6 was initially described as tubulin deacetylase; however, it also deacetylates other non-histone proteins, including heat-shock protein (Hsp90). Importantly, HDAC6 has an essential role in the aggresomal protein degradation pathway, an alternative system to the proteasome for degradation of polyubiquitinated misfolded/unfolded proteins. Specifically, HDAC6 binds to both polyubiquitinated proteins and dynein motors, thereby acting to recruit protein cargo to dynein motors for transport to aggresomes for lysosomal degradation.1 We have previously shown that inhibition of both proteasomal and aggresomal protein degradation using bortezomib (BTZ) to inhibit the proteasome and tubacin to inhibit HDAC6, respectively, induces accumulation of polyubiquitinated proteins and significant multiple myeloma (MM) cell stress, followed by apoptosis.2 More recently, a hydroxamic acid HDAC6-selective inhibitor ACY-1215 combined with BTZ shows significant anti-MM activities in preclinical studies,3 which have been rapidly translated to phase I/II clinical studies to treat relapsed/refractory MM.4 Previous studies have also shown that the non-selective HDAC inhibitor vorinostat in combination with BTZ also triggers synergistic cytotoxicity in MM,5 which has provided the framework for combination clinical trials.6 Carfilzomib (CFZ) is an epoxyketone proteasome inhibitor, which recently received accelerated Food and Drug Administration approval to treat relapsed refractory MM.7,8 In this study, we asked whether HDAC6 inhibitors enhance anti-MM toxicity induced by CFZ. We also delineated differential molecular mechanisms that trigger synergistic MM toxicity triggered by CFZ in combination with either HDAC6-selective or class-I HDAC-selective inhibitors ACY-1215 and MS275 (entinostat), respectively.9 We first compared the combination effect of the HDAC6-selective inhibitor ACY-1215 with either BTZ or CFZ in MM cell lines. Synergistic cytotoxicity induced by CFZ with ACY-1215 was greater than that by BTZ with ACY-1215 in both RPMI8226 (Figure 1a, upper panel) and MM.1S cells (Figure 1a, lower panel). To further confirm the role of HDAC6 inhibition, we carried out similar combination cytotoxicity experiments using the chemically
unrelated HDAC6-selective inhibitor tubastatin-A. Consistent with the ACY-1215 results, tubastatin-A synergistically enhanced both BTZ- and CFZ-induced cytotoxicity. Interestingly, synergistic cytotoxicity, on the basis of combination index (CI) o1, was more significant in RPMI8226 cells than in MM.1S cells (Supplementary Figure 1). Although BTZ has a remarkable clinical activity in MM, acquired resistance limits its long-term utility. We, therefore, next examined whether HDAC6 inhibition can overcome BTZ resistance in vitro using the BTZ-resistant ANBL (ANBL-R) human MM cell line. Importantly, the synergistic cytotoxicity of ACY-1215 with BTZ was observed even in ANBL-R cells. Similar to Figure 1a, this synergistic cytotoxicity was even more significant with CFZ compared with BTZ (Figure 1b). These results suggest that CFZ may have a greater clinical activity in MM than in BTZ when combined with HDAC6 inhibitors. We and others have shown that non-selective HDAC inhibitors in combination with BTZ trigger synergistic cytotoxicity in MM.10,11 These non-selective HDAC inhibitors block not only HDAC6 but also the class-I HDAC activity,9 and whether synergistic cytotoxicity induced by non-selective HDAC inhibitors with BTZ is due solely to HDAC6 blockade remains unclear. Indeed, we have recently shown that both HDAC3 knockdown and a smallmolecule HDAC3-selective inhibitor BG45 trigger synergistic MM cytotoxicity in combination with BTZ.12 To address this question, we first examined the cytotoxicity of class-I HDAC-selective inhibitor MS275 in combination with CFZ. MS275 with CFZ induced synergistic cytotoxicity in RPMI8226 cells and additive cytotoxicity in MM.1S cells (Supplementary Figure 2). Consistent with our studies, MS275 with BTZ also shows significant antitumor activities in other cancer cell types.13 We next determined whether different molecular mechanisms mediate the synergistic MM cell cytotoxicity of HDAC6 versus class-I HDAC inhibition in combination with CFZ. We first defined the doses of either ACY-1215 (2 mM) or MS275 (2 mM) with CFZ (10 nM) that induced equivalent cytotoxicity (35%) (Supplementary Figure 3) and used these culture conditions for subsequent experiments. We have shown that ACY-1215 with BTZ triggers the accumulation of polyubiquitinated proteins associated with cell stress.3 Although not all polyubiquitinated proteins are degraded by the proteasome, recent studies have shown that proteins specifically linked with lysine (K)48 polyubiquitin are degraded by proteasomes.14 Our studies showed that CFZ induced
Accepted article preview online 22 October 2013; advance online publication, 12 November 2013
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Leukemia (2014) 404 – 463
