Session: 604. Molecular Pharmacology and Drug Resistance: Myeloid Neoplasms: Poster II
Hematology Disease Topics & Pathways:
Research, Acute Myeloid Malignancies, AML, Adult, Fundamental Science, Translational Research, Hematopoiesis, Diseases, Myeloid Malignancies, Biological Processes, Molecular biology, Study Population, Human, Animal model, Pathogenesis
Methods: In murine retrovirally-induced AML cells (Hoxa9-Meis1, H9M; MN1) treated with LSD1 inhibitor (Bomedemstat) and ATRA, we assessed proliferation via the methoxynitrosulfophenyl-tetrazolium carboxanilide (XTT) assay, and the differentiation effect by flow cytometry. Gene expression levels of key myeloid transcription factors (TFs) and targets of ATRA (Spi1, Gfi1/1b, Rara, Tgm2) were determined via quantitative PCR. We generated H9M-transformed myeloid progenitor cells on an Irf8 knockout (KO) background, each line engrafted and generated AML.
Results: H9M wild-type (WT) cells had an IC50 for ATRA in the nanomolar range (47 nM) which increased 151-fold to the micromolar range (7.1 µM) in H9M Irf8 KO (p≤ 0.05). The IC50 for Bomedemstat also increased 9.7-fold in the Irf8 KO (p≤ 0.05).
While both H9M WT and H9M Irf8 KO demonstrated a reduction in the proportion of c-Kit+ cells, the upregulation of CD11b was significantly dampened in H9M Irf8 KO population, leading to a reduction in CD11b mean fluorescence intensity by 2.1-fold after treatment (p≤0.0001).
To determine the relationship between IRF8 expression and the sensitivity of leukemic cells to ATRA, we examined the effect of the Irf8 KO on the expression of Rara and the regulation of RARA targets upon treatment. Based on the published data (Oki, S; Ohta, T (2015): ChIP-Atlas) we identified several direct binding sites for IRF8 in key regulatory regions of Rara. When comparing the expression of Rara between H9M and H9M Irf8 KO we found a 2.1-fold lower expression of Rara in Irf8 KO cells. Consistently, H9M Irf8 KO showed decreased responsiveness of an important ATRA target gene (Tgm2) upon treatment with ATRA.
We investigated changes in core myeloid transcription factors interacting with LSD1i by qPCR. H9M Irf8 KO had 2.35-fold higher expression of Gfi1 compared to H9M WT cells but showed 26-fold lower expression of Gfi1b at baseline. Upon treatment with Bomedemstat, both H9M and H9M Irf8 KO cells showed a decrease in Gfi1 expression by 4.6 / 1.7-fold and a strong upregulation of Gfi1b by 38-fold / 460-fold, respectively. However, we observed a differential regulation of Gata1 and Gata2 between the models. While Gata1 expression is increased after treatment with Bomedemstat in H9M WT cells, it decreases in H9M Irf8 KO. Gata2 is showing the opposite trend consistent with the mutual regulation of these transcription factors.
Conclusion: In summary, we found that IRF8 expression modulates the response of AML cells to both LSD1 inhibition and ATRA. These findings suggest IRF8 expression is a potential biomarker for selecting patients for treatment with an LSD1 inhibitor and ATRA.
Disclosures: Khalaf: Novartis: Honoraria; Pfizer: Honoraria; Paladin: Honoraria. Garcia-Horton: Bristol Myers Squibb: Honoraria. Foley: Novartis and Gilead: Other: Advisory boards ; Novartis, Gilead and Janssen: Honoraria; Gilead, Novartis, Celgene: Speakers Bureau. Leber: SOBI: Research Funding, Speakers Bureau; Alexion/GSK: Research Funding, Speakers Bureau; Treadwell: Research Funding, Speakers Bureau; Jazz: Research Funding, Speakers Bureau; Astellas: Research Funding, Speakers Bureau; Amgen: Research Funding, Speakers Bureau; Novartis: Research Funding, Speakers Bureau; Pfizer: Research Funding, Speakers Bureau; Abbvie: Consultancy, Research Funding, Speakers Bureau; Otsuka: Research Funding, Speakers Bureau; Celgene: Research Funding, Speakers Bureau; BMS: Research Funding, Speakers Bureau; Astex: Research Funding, Speakers Bureau; Paladin: Research Funding, Speakers Bureau; Roche: Research Funding, Speakers Bureau; Servier: Research Funding, Speakers Bureau; Janssen: Research Funding, Speakers Bureau. Walker: Sanofi: Membership on an entity's Board of Directors or advisory committees, Research Funding. Kleppe: Merck: Ended employment in the past 24 months. Rienhoff: Merck: Ended employment in the past 24 months. Berg: Jazz Pharmaceuticals: Honoraria; BMS: Honoraria; AVIR Pharma: Honoraria; Imago Biosciences (a subsidiary of Merck): Research Funding.
See more of: Oral and Poster Abstracts