EHA Library - The official digital education library of European Hematology Association (EHA)

NUCLEOSOME BINDING PROTEIN HMGN1 BLOCKS MYELOID DIFFERENTIATION AND PROMOTES CLONAL DOMINANCE VIA ABERRANT HISTONE ACETYLATION
Author(s): ,
Lucia Cabal-Hierro
Affiliations:
Dana Farber Cancer Insitute/ Harvard Medical School,Boston,United States;Broad Institute,Cambridge,United States
,
Peter Van Galen
Affiliations:
Massachusetts General Hospital,Boston,United States;Broad Institute,Cambridge,United States
,
Miguel Prado
Affiliations:
Harvard Medical School,Boston,United States
,
Katsuhiro Togami
Affiliations:
Dana Farber Cancer Insitute/ Harvard Medical School,Boston,United States;Broad Institute,Cambridge,United States
,
Cody Mowery
Affiliations:
Dana Farber Cancer Insitute/ Harvard Medical School,Boston,United States;Broad Institute,Cambridge,United States
,
Kelly Higby
Affiliations:
Dana Farber Cancer Insitute/ Harvard Medical School,Boston,United States;Broad Institute,Cambridge,United States
,
David Sykes
Affiliations:
Massachusetts General Hospital,Boston,United States
,
Steven Gygi
Affiliations:
Harvard Medical School,Boston,United States
,
Bradley Bernstein
Affiliations:
Massachusetts General Hospital,Boston,United States;Broad Institute,Cambridge,United States
Andrew Lane
Affiliations:
Dana Farber Cancer Insitute/ Harvard Medical School,Boston,United States;Broad Institute,Cambridge,United States
(Abstract release date: 05/18/17) EHA Library. Cabal-Hierro L. 06/24/17; 181715; S428
Lucia Cabal-Hierro
Lucia Cabal-Hierro
Contributions
Abstract

Abstract: S428

Type: Oral Presentation

Presentation during EHA22: On Saturday, June 24, 2017 from 11:45 - 12:00

Location: Hall E

Background

Acute myeloid leukemia (AML) is characterized by rapid growth and block in differentiation of myeloid progenitors. The AML blast is defined by having “open” chromatin. We hypothesized that alterations of chromatin compaction may promote AML. Reversing those changes could represent a novel therapeutic approach.

Aims

Gain of chr21q22 is the most common focal amplification in complex karyotype AML. HMGN1 is a chromatin-regulatory protein on 21q22 known to affect lymphoid development, and our preliminary data suggested that HMGN1 could directly mediate a myeloid differentiation block. Since HMGN1 is known to decompact chromatin and alter histone marks, our goal was to define and therapeutically target the mechanisms by which HMGN1 overexpression disrupts myeloid differentiation and promotes clonal dominance

Methods

We immortalized bone marrow progenitors from wild-type (WT) or OE-HMGN1 mice (transgenic overexpressing HMGN1) with an estrogen receptor-HoxB8 fusion protein. Using exogenous estrogen to control nuclear translocation of HoxB8, we analyzed synchronized myeloid differentiation by flow cytometry, RNAseq, and TMT proteomic analysis. We performed MINT-ChIP-seq (MNase Indexed T7-chromatin IP) to measure the histone marks H3K27ac, H3K27me3, H3K4me3 and total Histone H3. We also measured histone marks in hematopoietic stem and progenitor subpopulations in vivo. We performed competitive bone marrow transplantation with CD45.1 WT and CD45.2 OE-HMGN1 donors and measured the relative contribution to hematopoiesis over time. 

Results

Synchronized differentiation in WT cells progressed over 6 days from myeloid progenitors to mature neutrophils and monocytes, analyzed by cell surface markers, morphology, and gene and protein expression. OE-HMGN1 cells proliferated faster and remained as undifferentiated myeloblasts (84% Cd11b+Gr1+ in WT vs 4% in OE-HMGN1, p=0.002; Fig A). Gene set enrichment analysis revealed more similarity to undifferentiated hematopoiesis and leukemia signatures in OE-HMGN1 cells. MINT-ChIP indicated higher global and locus-specific levels of H3K27ac in OE-HMGN1 cells (Fig B, upper panel), consistent with an increase in gene transcription, confirmed by RNA-seq. We found a specific increase in HoxA cluster expression in OE-HMGN1 cells, highest at HoxA7 and HoxA9, genes known to be important in AML pathogenesis. In agreement with gene expression, among the most differentially measured histone peaks genome-wide were higher H3K27ac at HoxA gene promoters at all differentiation time points analyzed (Fig B, lower panel). Competitive transplantation demonstrated an advantage to OE-HMGN1 stem and progenitor cells. The clonal dominance of OE-HMGN1 over WT cells extended to all populations analyzed (long- and short-term HSCs, multipotent progenitors, CMP, GMP and MEP; Fig C) and to mature lineages (myeloid, B and T cells). MINT-ChIP in LK and LKS stem and progenitor cells revealed an increase in H3K27ac peaks at cell cycle and leukemia-related genes in the context of OE-HMGN1. H3K27 acetylation is catalyzed by the CBP/p300 histone acetyltransferase (HAT), suggesting that HAT inhibition could target leukemias with HMGN1 overexpression. Indeed, treatment of myeloid progenitors with the CBP/p300 inhibitor C646 rescued the differentiation block in OE-HMGN1 cells (93% CD11b+Gr1+ in WT vs 80% in OE-HMGN1, p=NS).  

Conclusion

Our study suggests that HMGN1 overexpression blocks myeloid differentiation and promotes proliferation in hematopoietic progenitors via increased H3K27 acetylation. Targeting epigenetic changes downstream of HMGN1 or interfering with HMGN1 itself may represent a novel therapeutic strategy in AML
 

Session topic: 3. Acute myeloid leukemia - Biology

Keyword(s): Histone acetylation, Chromatin, AML

Abstract: S428

Type: Oral Presentation

Presentation during EHA22: On Saturday, June 24, 2017 from 11:45 - 12:00

Location: Hall E

Background

Acute myeloid leukemia (AML) is characterized by rapid growth and block in differentiation of myeloid progenitors. The AML blast is defined by having “open” chromatin. We hypothesized that alterations of chromatin compaction may promote AML. Reversing those changes could represent a novel therapeutic approach.

Aims

Gain of chr21q22 is the most common focal amplification in complex karyotype AML. HMGN1 is a chromatin-regulatory protein on 21q22 known to affect lymphoid development, and our preliminary data suggested that HMGN1 could directly mediate a myeloid differentiation block. Since HMGN1 is known to decompact chromatin and alter histone marks, our goal was to define and therapeutically target the mechanisms by which HMGN1 overexpression disrupts myeloid differentiation and promotes clonal dominance

Methods

We immortalized bone marrow progenitors from wild-type (WT) or OE-HMGN1 mice (transgenic overexpressing HMGN1) with an estrogen receptor-HoxB8 fusion protein. Using exogenous estrogen to control nuclear translocation of HoxB8, we analyzed synchronized myeloid differentiation by flow cytometry, RNAseq, and TMT proteomic analysis. We performed MINT-ChIP-seq (MNase Indexed T7-chromatin IP) to measure the histone marks H3K27ac, H3K27me3, H3K4me3 and total Histone H3. We also measured histone marks in hematopoietic stem and progenitor subpopulations in vivo. We performed competitive bone marrow transplantation with CD45.1 WT and CD45.2 OE-HMGN1 donors and measured the relative contribution to hematopoiesis over time. 

Results

Synchronized differentiation in WT cells progressed over 6 days from myeloid progenitors to mature neutrophils and monocytes, analyzed by cell surface markers, morphology, and gene and protein expression. OE-HMGN1 cells proliferated faster and remained as undifferentiated myeloblasts (84% Cd11b+Gr1+ in WT vs 4% in OE-HMGN1, p=0.002; Fig A). Gene set enrichment analysis revealed more similarity to undifferentiated hematopoiesis and leukemia signatures in OE-HMGN1 cells. MINT-ChIP indicated higher global and locus-specific levels of H3K27ac in OE-HMGN1 cells (Fig B, upper panel), consistent with an increase in gene transcription, confirmed by RNA-seq. We found a specific increase in HoxA cluster expression in OE-HMGN1 cells, highest at HoxA7 and HoxA9, genes known to be important in AML pathogenesis. In agreement with gene expression, among the most differentially measured histone peaks genome-wide were higher H3K27ac at HoxA gene promoters at all differentiation time points analyzed (Fig B, lower panel). Competitive transplantation demonstrated an advantage to OE-HMGN1 stem and progenitor cells. The clonal dominance of OE-HMGN1 over WT cells extended to all populations analyzed (long- and short-term HSCs, multipotent progenitors, CMP, GMP and MEP; Fig C) and to mature lineages (myeloid, B and T cells). MINT-ChIP in LK and LKS stem and progenitor cells revealed an increase in H3K27ac peaks at cell cycle and leukemia-related genes in the context of OE-HMGN1. H3K27 acetylation is catalyzed by the CBP/p300 histone acetyltransferase (HAT), suggesting that HAT inhibition could target leukemias with HMGN1 overexpression. Indeed, treatment of myeloid progenitors with the CBP/p300 inhibitor C646 rescued the differentiation block in OE-HMGN1 cells (93% CD11b+Gr1+ in WT vs 80% in OE-HMGN1, p=NS).  

Conclusion

Our study suggests that HMGN1 overexpression blocks myeloid differentiation and promotes proliferation in hematopoietic progenitors via increased H3K27 acetylation. Targeting epigenetic changes downstream of HMGN1 or interfering with HMGN1 itself may represent a novel therapeutic strategy in AML
 

Session topic: 3. Acute myeloid leukemia - Biology

Keyword(s): Histone acetylation, Chromatin, AML

By clicking “Accept Terms & all Cookies” or by continuing to browse, you agree to the storing of third-party cookies on your device to enhance your user experience and agree to the user terms and conditions of this learning management system (LMS).

Cookie Settings
Accept Terms & all Cookies