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Combined LSD1 and ATRA in AML
gene expression through demethylation of mono and dimethyl groups present on lysine 4 of histone H3.6 LSD1 is a critical regulator of hematopoiesis, in part, through interaction with the transcription factors GFI-1 and GFI- 1b. This LSD1-containing complex regulates expression of key myeloid differentiation genes and ultimately con- trols hematopoietic progenitor cell differentiation.7 LSD1 is frequently over-expressed in human cancers, including AML, and knockdown of LSD1 has been shown to inhibit the growth of AML cells.1,8-10 These data have spurred interest in LSD1 as a potential target for treatment of AML. As previously reported, potent, selective, irre- versible inactivators of LSD1 have been developed, and among the cancer cell lines evaluated, these show selec- tive anti-proliferative activity in SCLC and AML cell lines.9,11-13 Preclinical data such as these have led to the clinical development of LSD1 inhibitors in relapsed, refractory AML patients.
To build upon the therapeutic potential of LSD1 inhibi- tion in AML, rational combination hypotheses and com- binations with standard of care agents were considered. All-trans retinoic acid (ATRA) is used clinically to treat acute promyelocytic leukemia (APL), a subtype of AML, and has been shown to be hugely successful, achieving curative effects in this disease subtype.14 ATRA triggers the transcription factor retinoic acid receptor alpha (RARα) to bind to retinoic acid response elements found in the genome and initiate transcription of target genes, including those important for differentiation.15 APL is characterized by a PML-RARα fusion that inactivates RARα by preventing it from its normal binding and thus locking the tumor in an undifferentiated state. ATRA degrades this fusion, allowing RARα to activate its target genes, leading to differentiation and apoptosis of the can- cer cells.16,17 Many clinical trials have attempted to extend the use of ATRA into non-APL AML, but unfortunately these have demonstrated very little success.18 Since the discovery of LSD1 and the characterization of its role in hematopoiesis, there has been speculation as to the pos- sibility of combining an inhibitor of LSD1 with ATRA. One report demonstrated that combination of ATRA with knockdown of LSD1 or tranylcypromine, a non- selective monoamine oxidase inhibitor with weak LSD1 inhibitory activity, leads to transcriptional activation of many RAR target genes that normally lack methylation of H3K4me2 at their promoters.19,20 This combination also had more robust anti-leukemic activity than either treat- ment alone in the model tested.19
The current report demonstrates the synergistic activity of a combination of a selective, potent inhibitor of LSD1, GSK2879552, with ATRA, and characterizes the mecha- nism associated with this combination. As a single agent, LSD1 inhibition promotes differentiation of AML cell lines and synergistic differentiation activity is observed when used in combination with ATRA across AML sub- types. This combination also enhances LSD1 inhibitor- mediated growth inhibition of AML cell lines and pri- mary patient samples. Most importantly, treatment of AML cell lines with an LSD1 inhibitor and ATRA results in synergistic cytotoxicity and caspase-mediated cell death. Collectively, these data suggest that this combina- tion may result in greater efficacy and may be more likely to translate to response in AML patients than LSD1 inhibitor monotherapy. Moreover, this combination may expand the therapeutic potential of ATRA, a proven ther-
apy, to non-APL AML patients. Clinical studies are cur- rently underway to test this hypothesis in relapsed, refractory AML patients (clinicaltrials.gov identifiers: 02273102, 02717884, 02842827).
Methods
Cell lines and human biological samples
Cell lines were obtained from the American Type Culture Collection (ATCC) or the Deutsche Sammlung von Mikroorganismen und Zellbulturen (DSMZ). Human biological samples were sourced ethically and informed consent was obtained for their use in research. The use of human tissue sam- ples was reviewed and approved by the GSK Research & Development Compliance (RDC) Human Biological Sample Use Committee.
Compounds
GSK2879552 and GSK-LSD1 (GlaxoSmithKline), ATRA (Sigma), and Bortezomib (Chemietek) were prepared in 100% dimethyl sulfoxide (DMSO) (Sigma).
Cell proliferation
Proliferation assays were conducted as previously described.11 The growth-death index (GDI) value was determined as the per- centage of cells relative to end-of-assay vehicle and assay start, where the number of cells in the vehicle is set at 100% and the number of cells at the time of compound addition (T0) is set to 0%. A minimum of two biological replicates were evaluated for each assay. Bliss Independence analysis was performed, and a synergy score was determined.9,21
Mechanistic and phenotypic assays
A minimum of two biological replicates were evaluated for each assay. Dose response curves were generated using a 4-parameter equation (XLfit, IDBS). All kits were used according to the manu- facturer’s recommendations. Cell cycle phase distribution was determined by flow cytometry using the CycletestTM PLUS DNA Reagent Kit (BD Biosciences) and data were analyzed using FlowJo software (TreeStar Inc.). Proliferation was assessed using BrdU Cell Proliferation Assay Kit (Cell Signaling Technology) with a 4-hour pulse. Values were expressed as percent of vehicle. Caspase activity was measured using Caspase-Glo 3/7 (Promega). Luminescence values were normalized to CTG (Promega) for cell number. Peak activation was determined for MOLM-13, OCI- AML3, MV-4-11, THP-1, SIG-M5, HL-60, and Kasumi-1 to be days 4, 6, 6, 3, 4, 5, and 5, respectively. Values were expressed as a fold change relative to vehicle. Gene expression was evaluated using real time-quantitative polymerase chain reaction (RT-qPCR), as previously described.11 Superoxide anion production was meas- ured using LumiMax Superoxide Anion Detection Kit (Agilent Technologies). Values were expressed as fold increase over vehi- cle. Morphology was visually assessed after staining with May- Grunwald and Giemsa (Sigma).
Flow cytometry
Cells were stained with surface marker antibodies (CD11b, 555388 or 557754; CD86, 555657; CD71, 555537) and 7-AAD obtained from BD Biosciences. Percent positive for each marker was determined relative to isotype control from cells that were 7- AAD negative. Additionally, cells were stained with the above cocktail including annexin V (BD Biosciences) to evaluate Annexin V positivity. A minimum of two biological replicates were evalu- ated for each assay.
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