IDENTIFICATION AND TARGETING OF MOLECULAR SIGNATURES OF HYPOMETHYLATING AGENT RESISTANCE IN MYELODYSPLASTIC SYNDROME AND CHRONIC MYELOMONOCYTIC LEUKEMIA
Author(s): ,
Yue Wei
Affiliations:
Leukemia,M D Anderson Cancer Center,Houston,United States
,
Caleb Class
Affiliations:
Biostatistics,M D Anderson Cancer Center,Houston,United States
,
Rashmi Kanagal-Shamanna
Affiliations:
Hematopathology,M D Anderson Cancer Center,Houston,United States
,
Hong Zheng
Affiliations:
Leukemia,M D Anderson Cancer Center,Houston,United States
,
Marcos Estecio
Affiliations:
Epigenetics and Molecular Carcinogenesis,M D Anderson Cancer Center,Houston,United States
,
Yue Lu
Affiliations:
Epigenetics and Molecular Carcinogenesis,M D Anderson Cancer Center,Houston,United States
,
Kelly Soltysiak
Affiliations:
Leukemia,M D Anderson Cancer Center,Houston,United States
,
Hui Yang
Affiliations:
Leukemia,M D Anderson Cancer Center,Houston,United States
,
Koji Sasaki
Affiliations:
Leukemia,M D Anderson Cancer Center,Houston,United States
,
Guillermo Montalban-Bravo
Affiliations:
Leukemia,M D Anderson Cancer Center,Houston,United States
,
Irene Ganan-Gomez
Affiliations:
Leukemia,M D Anderson Cancer Center,Houston,United States
,
Kim-Anh Do
Affiliations:
Biostatistics,M D Anderson Cancer Center,Houston,United States
,
Simona Colla
Affiliations:
Leukemia,M D Anderson Cancer Center,Houston,United States
,
Carlos Bueso-Ramos
Affiliations:
Hematopathology,M D Anderson Cancer Center,Houston,United States
Guillermo Garcia-Manero
Affiliations:
Leukemia,M D Anderson Cancer Center,Houston,United States
EHA Library. Wei Y. Jun 15, 2019; 266942; PS1325
Dr. Yue Wei
Dr. Yue Wei
Contributions
Abstract

Abstract: PS1325

Type: Poster Presentation

Presentation during EHA24: On Saturday, June 15, 2019 from 17:30 - 19:00

Location: Poster area

Background
Hypomethylating agent (HMA) failure has significant adverse outcomes in MDS and CMML but still without available biomarkers and licensed second-line treatments. 

Aims
We aimed to better understand the biology underlying HMA failure via in-depth comparison of DNA methylation, genetic mutation, and RNA expression between HMA responders and non-responders in patients. 

Methods
Baseline BM aspirates were collected from 64 patients with MDS (N=50) and CMML (N=14) before HMA therapy. There were 37 HMA responders and 27 non-responders. DNA methylation was analyzed in BM mononuclear cells (MNCs) using reduced representation bisulfied sequencing (RRBS). Differentially methylated regions (DMR) were identified using methylKit and edmr R, and were defined as mean methylation difference of 15% and at least 1 base with 25% difference. Targeted next-generation sequencing (NGS) mutation analysis was performed on the MNCs using an 81-gene panel. A minimum of 250x bidirectional coverage with a minimum of 2% allelic burden was required for variant calling. Whole Exome RNA-Seq was performed in CD34+ BM hematopoietic stem and progenitor cells (HSPCs). FASTQ files were processed in TopHat2. Differential gene expression analysis was conducted using DESeq2. Pathway analysis was performed using gene set enrichment analysis (GSEA), with the fgsea library in R and 10,000 gene permutation. A false discovery corrected p-value of 0.1 was required.

Results
Slightly higher DNA methylation was identified in non-responders with 55 DMRs mostly located in CpG islands. DMRs in 3 genes (HTR1AGRM1, and ST8SIA3) were identified, and were hypermethylated in non-responders. This result suggests that HMA response-associated baseline DMR is rare.

NGS idendified that ASXL1 mutation (47% in non-responders vs 14% in responders) and NRAS mutation (20% vs 0%) were significantly associated with poor response (p<0.05), and NRAS mutation was concomitant with ASXL1 mutation. ASXL1 mutation was also associated with hyper-activation of MAPK and NF-kB signaling in BM cells (p<0.05, q<0.05).   

RNA-Seq on BM CD34+ cells revealed 29 genes differentially expressed between responders and non-responders (q<0.1). GSEA indicated that type I interferon and neurotransmitter signals were significantly up-regulated in non-responders. In MDS cohort, the RIG-like receptor (RLR) signaling, an IFN signal activating pattern recognition receptor pathway, was significantly elevated in non-responders. mRNA expression of RLR3 was higher in MDS non-responders versus responders (p<0.05, q>0.05).

The finding in patients was validated in an HMA-resistant TF1-RES cell line: Based on RNA-Seq, IFN signaling was upregulated in TF1-RES compared to HMA-sensitive TF1 cells (p<0.05, q<0.05), and was associated with increased expression of RLR2 gene (p<0.05, q<0.05). RNA inhibition of RLR2 in TF1-RES reduced expression of IFN signal gene and sensitized cells to HMA. RLR-mediated IFN signaling activation is known to crosstalk with oxidative phosphorylation (OXPHOS). We observed increased OXPHOS in TF1-RES compared to TF1 cells. IACS10759, a clinical-grade OXPHOS inhibitor, led to decreased expression of IFN signaling genes and increased HMA sensitivity in TF1-RES cell. In patient BM CD34+ cells who failed HMA, IACS10759 also increased their sensitivity to HMA.

Conclusion
ASXL1 mutation and hyper-activated IFN signal were identified as potential biomarkers of HMA resistance in MDS and CMML. Targeting IFN-RLR-OXPHOS activity has therapeutic potential to circumvent HMA failure. 

Session topic: 9. Myelodysplastic syndromes - Biology & Translational Research

Keyword(s): Drug resistance, Hypomethylation, Myelodysplasia

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