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J. Stomper et al.
With the perspective of exploiting HbF as a potential biomarker for response to HMA therapy, it might be used for selecting a cohort of MDS/AML patients who exhibit a significant increase in HbF after two cycles of decitabine treatment as candidates for a future clinical trial. Such a trial could have the objective to increase the effectiveness of HMA therapy by dose-schedule modifications and/or in combination with other epigenetic or other agents to increase the duration of response.
In practical terms, HbF measurement during HMA treatment needs to be compared in different laboratories (at this study’s central laboratory, HPLC quantification of different rare Hb species, including HbF, has been opti- mized for linear quantification also of smaller amounts). However, baseline HbF is also a promising predictor of HMA response and outcome, as recently demonstrated in the same cohorts of patients.16 At first sight it appears counter-intuitive that pre-treatment high expression may be biologically linked to outcome. However, as already discussed,16 elevated pre-treatment HbF levels may reflect incomplete silencing by methylation (and possibly other epigenetic mechanisms) of the gamma-globin locus, pro- viding a potential surrogate marker for higher de novo sen- sitivity to HMAs. Interestingly, Cross et al.32 made a simi- lar observation studying long interspersed element (LINE)-1 methylation in AML patients treated with azac- itidine: lower LINE-1 methylation prior to treatment, but not early hypomethylation under treatment, predicted hematologic response to this in vivo hypomethylation.
What is the cellular source of HbF during the different treatment phases in MDS and AML patients receiving HMAs? In untreated MDS patients clonal erythroid pro- genitors,33 and in untreated AML patients residual normal, non-clonal erythroid progenitors may be the source of HbF-containing erythrocytes.34 In contrast, in patients attaining a complete or cytogenetic remission, it is more likely that the target cell of HMAs (presumably achieving hypomethylation and transcriptional de-repression of the beta-globin-like gene locus) is part of the non-clonal ery- thropoiesis, as in patients with solid tumors who show HbF induction during HMA treatment.10,11 The increase in platelet count after decitabine treatment indicates that decitabine reduced or eliminated the suppressive action of the malignant cells on normal hematopoiesis, with subsequent expansion of normal hematopoietic stem cells, which undergo differentiation. At the time of hema- tologic relapse, the decline in HbF levels might be due to the recurrence of the malignant clone. This contrasts with the model of increased HbF levels in juvenile myelomonocytic leukemia resulting from epigenetic dys- regulation of beta-like-globin genes in leukemic cells.35 Serial immunohistochemical bone marrow studies for HbF expression in MDS/AML patients receiving HMAs are warranted to determine the cell of origin of HbF pro- duction during the different phases of treatment.
Modeling the effects of decitabine on the erythroid ver- sus megakaryocytic lineage in two bi-potential myeloid cell lines, activation of an erythroid but not megakary- ocytic gene expression program was observed, including induction of gamma-globin expression and induction, albeit modest, of HbF tetramer formation. We could demonstrate that decitabine treatment regulated many of the genes also regulated by hemin (including mRNA for erythroid-specific transcription factors and beta-like-glo- bin genes), and induced GATA1 at the protein level, con-
comitantly with demethylation at several cis-regulatory regions known to be important for the regulation of this gene.36,37 Notably, the overlap between the transcriptome changes induced by decitabine versus hemin was more marked in the downregulated genes compared to the upregulated ones. Despite demonstrating GATA1 gene demethylation following decitabine treatment, we are unable to conclude that GATA1 induction is a direct con- sequence of demethylation or is occurring during ery- throid differentiation triggered via other factors. Here, a similar DNA methylation analysis of K562 cells treated with hemin instead of decitabine would address this "cause or consequence" question.
Taken together, the cell line experiments suggested that increased levels of HbF can also occur because of the effects of decitabine on malignant cells of the erythroid lineage. However, since a cell line is not a good model of the normal functional hematopoietic hierarchy, no con- clusion can be drawn as to why erythroid rather than megakaryocytic differentiation was observed.
Is there a clinical relevance of induction of HbF beyond HMA treatment? Very recently, in a preclinical study a novel, specific inhibitor of histone deacetylase 1/2 also demonstrated a strong propensity to induce HbF.38 Furthermore, it is well established that inhibitors of the first histone lysine-specific demethylase (LSD)1, includ- ing novel, highly specific LSD1/KDM1A inhibitors such as RN-1, are able to reactivate a silenced beta-globin-like gene locus.39 Furthermore, UNC0638, a selective inhibitor of the histone methyltransferases EHMT1 and EHM2, has the ability to induce HbF expression, and this potency is enhanced when the drug is combined with decitabine or the histone deacetylase inhibitor entinostat.40 Very recently, pomalidomide was also shown to be able to induce HbF in patients with multiple myeloma.41 Thus, serial HbF measurements in these different clinical set- tings may be interesting in order to determine whether the kinetics of this parameter is predictive of treatment response.
In conclusion, the technically simple test of assaying HbF levels warrants further prospective studies since the time to best response in patients treated with HMAs is often in the range of 4-6 months. Earlier tailoring of treat- ment is, therefore, highly desirable. It will be of interest to determine the predictive value of HbF levels compared to other, already established predictors of HMA response such as hematologic parameters, genetic and DNA methylation markers.
Acknowledgments
The authors wish to thank Tobias Berg, Dietmar Pfeifer, Heike Pahl, Claudia Schmoor, Roland Schüle, Christian Flotho and others who kindly provided helpful input and critical discus- sion during the course of this study. We really appreciate the experimental contributions of Lena Pados (née Kasten), and the efforts of Thomas Epting (hemoglobin quantification) and Ljudmila Bogatyreva (statistics).
Funding
This work was supported by the DFG, SFB 992 (MEDEP, project C04). Further research funding: DFG-SPP 1463 (ML, CP), DFG-FOR 2674 (A01: CP; A05: ML/HB; A09: CP/ML), Wilhelm-Sander-Stiftung (grant 1999.032.2), German Cancer Aid (DKH 111210: HB; Max Eder stipend DKH 110461: RC).
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haematologica | 2019; 104(1)