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

MOLECULAR DETERMINANTS OF DISEASE PROGRESSION AFTER HYPOMETHYLATING AGENT THERAPY IN RAS PATHWAY MUTANT CHRONIC MYELOMONOCYTIC LEUKEMIA AT THE SINGLE-CELL LEVEL
Author(s): ,
Guillermo Montalban-Bravo
Affiliations:
Leukemia,The University of Texas MD Anderson Cancer Center,Houston,États-unis;Leukemia,The University of Texas MD Anderson Cancer Center,Houston,Vereinigte Staaten;Leukemia,The University of Texas MD Anderson Cancer Center,Houston,Stati Uniti;Leukemia,The University of Texas MD Anderson Cancer Center,Houston,United States;Leukemia,The University of Texas MD Anderson Cancer Center,Houston,Estados U
,
Feiyang Ma
Affiliations:
Molecular Biology Institute,University of California,Los Angeles,États-unis;Molecular Biology Institute,University of California,Los Angeles,Vereinigte Staaten;Molecular Biology Institute,University of California,Los Angeles,Stati Uniti;Molecular Biology Institute,University of California,Los Angeles,United States;Molecular Biology Institute,University of California,Los Angeles,Estados Unidos;Mole
,
Irene Ganan-Gomez
Affiliations:
Leukemia,The University of Texas MD Anderson Cancer Center,Houston,États-unis;Leukemia,The University of Texas MD Anderson Cancer Center,Houston,Vereinigte Staaten;Leukemia,The University of Texas MD Anderson Cancer Center,Houston,Stati Uniti;Leukemia,The University of Texas MD Anderson Cancer Center,Houston,United States;Leukemia,The University of Texas MD Anderson Cancer Center,Houston,Estados U
,
Rashmi Kanagal-Shamana
Affiliations:
Hematopathology,The University of Texas MD Anderson Cancer Center,Houston,États-unis;Hematopathology,The University of Texas MD Anderson Cancer Center,Houston,Vereinigte Staaten;Hematopathology,The University of Texas MD Anderson Cancer Center,Houston,Stati Uniti;Hematopathology,The University of Texas MD Anderson Cancer Center,Houston,United States;Hematopathology,The University of Texas MD Ander
,
Vera Adema
Affiliations:
Leukemia,The University of Texas MD Anderson Cancer Center,Houston,États-unis;Leukemia,The University of Texas MD Anderson Cancer Center,Houston,Vereinigte Staaten;Leukemia,The University of Texas MD Anderson Cancer Center,Houston,Stati Uniti;Leukemia,The University of Texas MD Anderson Cancer Center,Houston,United States;Leukemia,The University of Texas MD Anderson Cancer Center,Houston,Estados U
,
Natthakan Thongon
Affiliations:
Leukemia,The University of Texas MD Anderson Cancer Center,Houston,États-unis;Leukemia,The University of Texas MD Anderson Cancer Center,Houston,Vereinigte Staaten;Leukemia,The University of Texas MD Anderson Cancer Center,Houston,Stati Uniti;Leukemia,The University of Texas MD Anderson Cancer Center,Houston,United States;Leukemia,The University of Texas MD Anderson Cancer Center,Houston,Estados U
,
Hui Yang
Affiliations:
Leukemia,The University of Texas MD Anderson Cancer Center,Houston,États-unis;Leukemia,The University of Texas MD Anderson Cancer Center,Houston,Vereinigte Staaten;Leukemia,The University of Texas MD Anderson Cancer Center,Houston,Stati Uniti;Leukemia,The University of Texas MD Anderson Cancer Center,Houston,United States;Leukemia,The University of Texas MD Anderson Cancer Center,Houston,Estados U
,
Kelly A. Soltysiak
Affiliations:
Leukemia,The University of Texas MD Anderson Cancer Center,Houston,États-unis;Leukemia,The University of Texas MD Anderson Cancer Center,Houston,Vereinigte Staaten;Leukemia,The University of Texas MD Anderson Cancer Center,Houston,Stati Uniti;Leukemia,The University of Texas MD Anderson Cancer Center,Houston,United States;Leukemia,The University of Texas MD Anderson Cancer Center,Houston,Estados U
,
Carlos Bueso-Ramos
Affiliations:
Hematopathology,The University of Texas MD Anderson Cancer Center,Houston,États-unis;Hematopathology,The University of Texas MD Anderson Cancer Center,Houston,Vereinigte Staaten;Hematopathology,The University of Texas MD Anderson Cancer Center,Houston,Stati Uniti;Hematopathology,The University of Texas MD Anderson Cancer Center,Houston,United States;Hematopathology,The University of Texas MD Ander
,
Hagop Kantarjian
Affiliations:
Leukemia,The University of Texas MD Anderson Cancer Center,Houston,États-unis;Leukemia,The University of Texas MD Anderson Cancer Center,Houston,Vereinigte Staaten;Leukemia,The University of Texas MD Anderson Cancer Center,Houston,Stati Uniti;Leukemia,The University of Texas MD Anderson Cancer Center,Houston,United States;Leukemia,The University of Texas MD Anderson Cancer Center,Houston,Estados U
,
Guillermo Garcia-Manero
Affiliations:
Leukemia,The University of Texas MD Anderson Cancer Center,Houston,États-unis;Leukemia,The University of Texas MD Anderson Cancer Center,Houston,Vereinigte Staaten;Leukemia,The University of Texas MD Anderson Cancer Center,Houston,Stati Uniti;Leukemia,The University of Texas MD Anderson Cancer Center,Houston,United States;Leukemia,The University of Texas MD Anderson Cancer Center,Houston,Estados U
Simona Colla
Affiliations:
Leukemia,The University of Texas MD Anderson Cancer Center,Houston,États-unis;Leukemia,The University of Texas MD Anderson Cancer Center,Houston,Vereinigte Staaten;Leukemia,The University of Texas MD Anderson Cancer Center,Houston,Stati Uniti;Leukemia,The University of Texas MD Anderson Cancer Center,Houston,United States;Leukemia,The University of Texas MD Anderson Cancer Center,Houston,Estados U
(Abstract release date: 05/12/22) EHA Library. Montalban-Bravo G. 06/11/22; 357024; S160
Guillermo Montalban-Bravo
Guillermo Montalban-Bravo
Contributions
Abstract
Presentation during EHA2022: All Oral presentations will be presented between Friday, June 10 and Sunday, June 12 and will be accessible for on-demand viewing from Monday, June 20 until Monday, August 15, 2022 on the Congress platform.

Abstract: S160

Type: Oral Presentation

Session title: New insights in biology of bone marrow failure and MDS

Background

Most patients (pts) with chronic myelomonocytic leukemia (CMML) have incomplete or transient responses to hypomethylating agent (HMA) therapy. CMML cases driven by mutations in RAS pathway signaling genes or ASXL1 have a higher risk of failure and progression to acute myeloid leukemia (AML). Development of effective alternative therapies has been delayed, owing to an incomplete understanding of how different hematopoietic populations contributes to disease maintenance and progression.

Aims

We aimed to dissect the cellular and molecular mechanisms underpinning CMML maintenance and progression in RAS mutant CMML.

Methods

We performed single-cell RNA sequencing (scRNA-seq) analysis of lineage-negative (Lin-) CD34+ hematopoietic stem and progenitor cells (HSPCs) and BM mononuclear cells (MNCs) isolated from RAS pathway mutant CMML pts (n=5 and 6, respectively) and age-matched healthy donors (HD; n=2 and 3, respectively). CMML samples were obtained at the time of diagnosis and HMA failure. Additionally, we performed scATAC-seq analysis of Lin-CD34+ HPSCs isolated at the times of diagnosis and progression (n=1).

Results

Our analysis revealed that CMML HSPCs had a predominantly granulomonocytic differentiation route with increased frequencies of myeloid-monocytic progenitors, at the expense of hematopoietic stem cells (HSCs) (Fig 1a), and upregulated expression of genes involved in the oxidative phosphorylation, type I interferon (IFN) and IFNg pathways. Consistent with these results, scRNA-seq analysis of MNCs revealed expanded populations of myelomonocytic progenitors and monocytes and upregulated expression of genes involved in IFNg response and NF-kB activation (Fig 1b), along with upregulation of the NF-kB transcriptional effector BCL2A1 (Fig 1c). Assessment of ligand-receptor interactions using the CellPhoneDB repository identified that CMML monocytes established a high number of cell-cell interactions (n=638) with dendritic cells, NK cells, and HSPCs via chemokines, cytokines, and inhibitory molecules known to induce NF-kB signaling and NK-cell exhaustion. Disease progression was associated with expansion of lympho-myeloid progenitors (LMPPs) (Fig 1d) characterized by the highest levels of IFNg response, NF-kB survival signaling, and cell cycle regulators. scATAC-seq of Lin-CD34+ confirmed higher activity of transcriptional factors associated with monocytic differentiation and NF-kB signaling (Fig 1e-f). Accordingly, scRNA-seq analysis of MNCs showed increased frequencies of HSPCs and myelomonocytic precursors, a reduction of T cells (Fig 1f), and emergence of a monocyte population characterized by the highest expression of NF-kB signaling and its effectors MCL1 and BCL2A1. BCL2A1 protein expression at progression was confirmed by immunohistochemistry (Fig 1g). CellPhoneDB analysis identified a high number of cell-cell interactions (n=2978) involving cytokines, chemokines, and surface proteins known to elicit NF-kB activation and immune evasion between expanded monocytes, LMPPs, myelomonocytic precursors, and immune cells during progression.

Conclusion

Our data suggests that CMML is maintained through metabolically active HSPCs, which leads to monocytes’ reprograming and survival through NF-kB signaling activation. We showed that disease progression arises from the expansion of NF-kB dependent immature myeloid progenitors, which leads to therapy resistance and immune evasion. This study has implications for the development of therapies targeting downstream effectors of NF-kB–mediated survival pathway to overcome treatment failure. In vitro validation is ongoing.

Keyword(s): CMML, Progression, Ras, Stem and progenitor cell

Presentation during EHA2022: All Oral presentations will be presented between Friday, June 10 and Sunday, June 12 and will be accessible for on-demand viewing from Monday, June 20 until Monday, August 15, 2022 on the Congress platform.

Abstract: S160

Type: Oral Presentation

Session title: New insights in biology of bone marrow failure and MDS

Background

Most patients (pts) with chronic myelomonocytic leukemia (CMML) have incomplete or transient responses to hypomethylating agent (HMA) therapy. CMML cases driven by mutations in RAS pathway signaling genes or ASXL1 have a higher risk of failure and progression to acute myeloid leukemia (AML). Development of effective alternative therapies has been delayed, owing to an incomplete understanding of how different hematopoietic populations contributes to disease maintenance and progression.

Aims

We aimed to dissect the cellular and molecular mechanisms underpinning CMML maintenance and progression in RAS mutant CMML.

Methods

We performed single-cell RNA sequencing (scRNA-seq) analysis of lineage-negative (Lin-) CD34+ hematopoietic stem and progenitor cells (HSPCs) and BM mononuclear cells (MNCs) isolated from RAS pathway mutant CMML pts (n=5 and 6, respectively) and age-matched healthy donors (HD; n=2 and 3, respectively). CMML samples were obtained at the time of diagnosis and HMA failure. Additionally, we performed scATAC-seq analysis of Lin-CD34+ HPSCs isolated at the times of diagnosis and progression (n=1).

Results

Our analysis revealed that CMML HSPCs had a predominantly granulomonocytic differentiation route with increased frequencies of myeloid-monocytic progenitors, at the expense of hematopoietic stem cells (HSCs) (Fig 1a), and upregulated expression of genes involved in the oxidative phosphorylation, type I interferon (IFN) and IFNg pathways. Consistent with these results, scRNA-seq analysis of MNCs revealed expanded populations of myelomonocytic progenitors and monocytes and upregulated expression of genes involved in IFNg response and NF-kB activation (Fig 1b), along with upregulation of the NF-kB transcriptional effector BCL2A1 (Fig 1c). Assessment of ligand-receptor interactions using the CellPhoneDB repository identified that CMML monocytes established a high number of cell-cell interactions (n=638) with dendritic cells, NK cells, and HSPCs via chemokines, cytokines, and inhibitory molecules known to induce NF-kB signaling and NK-cell exhaustion. Disease progression was associated with expansion of lympho-myeloid progenitors (LMPPs) (Fig 1d) characterized by the highest levels of IFNg response, NF-kB survival signaling, and cell cycle regulators. scATAC-seq of Lin-CD34+ confirmed higher activity of transcriptional factors associated with monocytic differentiation and NF-kB signaling (Fig 1e-f). Accordingly, scRNA-seq analysis of MNCs showed increased frequencies of HSPCs and myelomonocytic precursors, a reduction of T cells (Fig 1f), and emergence of a monocyte population characterized by the highest expression of NF-kB signaling and its effectors MCL1 and BCL2A1. BCL2A1 protein expression at progression was confirmed by immunohistochemistry (Fig 1g). CellPhoneDB analysis identified a high number of cell-cell interactions (n=2978) involving cytokines, chemokines, and surface proteins known to elicit NF-kB activation and immune evasion between expanded monocytes, LMPPs, myelomonocytic precursors, and immune cells during progression.

Conclusion

Our data suggests that CMML is maintained through metabolically active HSPCs, which leads to monocytes’ reprograming and survival through NF-kB signaling activation. We showed that disease progression arises from the expansion of NF-kB dependent immature myeloid progenitors, which leads to therapy resistance and immune evasion. This study has implications for the development of therapies targeting downstream effectors of NF-kB–mediated survival pathway to overcome treatment failure. In vitro validation is ongoing.

Keyword(s): CMML, Progression, Ras, Stem and progenitor cell

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