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INHIBITION OF THE MYELOID MASTER REGULATOR PU.1 AS A THERAPEUTIC STRATEGY IN ACUTE MYELOID LEUKEMIA
Author(s):
Ileana Antony-Debre
,
Ileana Antony-Debre
Affiliations:
Albert Einstein Cancer Center,Albert Einstein College of Medicine,New York,United States;Inserm U1170,Gustave Roussy,Villejuif,France
Joana Leite
,
Joana Leite
Affiliations:
Albert Einstein Cancer Center,Albert Einstein College of Medicine,New York,United States
Ananya Paul
,
Ananya Paul
Affiliations:
Department of Chemistry,Georgia State University,Atlanta,United States
Kelly Mitchell
,
Kelly Mitchell
Affiliations:
Albert Einstein Cancer Center,Albert Einstein College of Medicine,New York,United States
Hye Mi Kim
,
Hye Mi Kim
Affiliations:
Department of Chemistry,Georgia State University,Atlanta,United States
Kenneth Huang
,
Kenneth Huang
Affiliations:
Department of Chemistry,Georgia State University,Atlanta,United States
Arvind Kumar
,
Arvind Kumar
Affiliations:
Department of Chemistry,Georgia State University,Atlanta,United States
Abdelbasset A Farahat
,
Abdelbasset A Farahat
Affiliations:
Department of Chemistry,Georgia State University,Atlanta,United States
Boris Bartholdy
,
Boris Bartholdy
Affiliations:
Albert Einstein Cancer Center,Albert Einstein College of Medicine,New York,United States
Swathi-Rao Narayanagari
,
Swathi-Rao Narayanagari
Affiliations:
Albert Einstein Cancer Center,Albert Einstein College of Medicine,New York,United States
Luis A Carvajal
,
Luis A Carvajal
Affiliations:
Albert Einstein Cancer Center,Albert Einstein College of Medicine,New York,United States
Jiahao Chen
,
Jiahao Chen
Affiliations:
Albert Einstein Cancer Center,Albert Einstein College of Medicine,New York,United States
Alberto Ambesi-Impiombato
,
Alberto Ambesi-Impiombato
Affiliations:
Institute for Cancer Genetics,Columbia University,New York,United States
Adolfo A Ferrando
,
Adolfo A Ferrando
Affiliations:
Institute for Cancer Genetics,Columbia University,New York,United States
Ioannis Mantzaris
,
Ioannis Mantzaris
Affiliations:
Albert Einstein Cancer Center,Albert Einstein College of Medicine,New York,United States
Evripidis Gavathiotis
,
Evripidis Gavathiotis
Affiliations:
Albert Einstein Cancer Center,Albert Einstein College of Medicine,New York,United States
Amit Verma
,
Amit Verma
Affiliations:
Albert Einstein Cancer Center,Albert Einstein College of Medicine,New York,United States
Britta Will
,
Britta Will
Affiliations:
Albert Einstein Cancer Center,Albert Einstein College of Medicine,New York,United States
David W Boykin
,
David W Boykin
Affiliations:
Department of Chemistry,Georgia State University,Atlanta,United States
W. David Wilson
,
W. David Wilson
Affiliations:
Department of Chemistry,Georgia State University,Atlanta,United States
Gregory M. K. Poon
,
Gregory M. K. Poon
Affiliations:
Department of Chemistry,Georgia State University,Atlanta,United States
Ulrich Steidl
Ulrich Steidl
Affiliations:
Albert Einstein Cancer Center,Albert Einstein College of Medicine,New York,United States
(Abstract release date: 05/18/17) EHA Library. Antony-Debre I. 06/23/17; 181422; S135
Ileana Antony-Debre
Ileana Antony-Debre
Contributions
PDF Abstract
Abstract

Abstract: S135

Type: Oral Presentation

Presentation during EHA22: On Friday, June 23, 2017 from 12:15 - 12:30

Location: Room N103

Background
Functionally critical decreases in PU.1 levels or activity are present across various different genetic and epigenetic subtypes of AML, and overall represent more than 50% of AML cases (Sive et al. Leukemia. 2016). However, approaches for the specific therapeutic targeting of these patients are thus far lacking. 

Aims
Retroviral restoration of PU.1 expression has previously been explored but is difficult to achieve pharmacologically. Here, we tested the inverse strategy. As complete loss of PU.1 leads to stem cell failure, we hypothesized that AML cells harboring already low levels of PU.1 may be more vulnerable to further PU.1 inhibition. 

Methods

We used two alternative approaches: RNA interference and newly developed small molecule PU.1 inhibitors. 

Results

We found that inhibition of PU.1 with different shRNAs led to a significant decrease in proliferation and clonogenicity, and increased apoptosis of mouse and human leukemic cell lines with low PU.1 levels, as well as the majority of primary human AML cells tested.
The pharmacologic targeting of transcription factor-DNA major groove interactions is challenging. However, specific PU.1 binding to chromatin critically depends on additional minor groove contacts upstream of the core ETS binding motif, which determine selectivity for PU.1. We used an integrated screening strategy utilizing biosensor surface plasmon resonance, DNA footprinting, and cell-based dual-color PU.1 reporter assays to develop novel small molecules of the heterocyclic diamidine family as first-in-class PU.1 inhibitors. Targeted occupancy by our compounds in the minor groove induces perturbations in DNA conformation that are transmitted to the PU.1 site in the major groove and thus inhibit PU.1 binding via an allosteric mechanism. Functionally, treatment with 3 different compounds decreased cell growth and colony forming capacity, increased apoptosis, and disrupted serial replating capacity of PU.1low AML cell lines, and a majority of primary AML cell samples. ChIP and expression analysis showed that the compounds disrupt PU.1-promoter interaction and lead to downregulation of canonical PU.1 transcriptional targets in AML cells, confirming on-target activity. Genome-wide analysis showed highly significant enrichment of known transcriptional targets of PU.1, and selectivity over other ETS family members. Comparison with published transcriptomic and PU.1 ChIP-seq data sets, as well as ARACNe analysis of the PU.1 regulon in primary AML cells, demonstrated that the inhibitors antagonized PU.1-regulated pathways at a genome-wide level. Treatment of normal HSPC in colony forming assays led to decreased production of mature granulo-monocytic cells, consistent with PU.1’s known role in this lineage. However, this effect was reversible upon drug removal, and serial replating capacity was not affected suggesting no significant effects on normal HSPC. Lastly, in vivo treatment with PU.1 inhibitors in mouse and human AML (xeno)transplantation models significantly decreased tumor burden and increased survival.

Conclusion

Our study describes for the first time a strategy inhibiting PU.1 in AML, establishing proof-of-concept for this approach. Furthermore, we report the development of first-in-class PU.1 inhibitors which interfere with PU.1-DNA interaction through an allosteric, minor groove-mediated mechanism. Our work shows that it is feasible to pharmacologically target PU.1, and raises intriguing possibilities for the potential targeting of other transcription factors through minor groove-directed approaches.  

Session topic: 3. Acute myeloid leukemia - Biology

Keyword(s): transcription factor, Targeted therapy, PU.1, Acute Myeloid Leukemia

Abstract: S135

Type: Oral Presentation

Presentation during EHA22: On Friday, June 23, 2017 from 12:15 - 12:30

Location: Room N103

Background
Functionally critical decreases in PU.1 levels or activity are present across various different genetic and epigenetic subtypes of AML, and overall represent more than 50% of AML cases (Sive et al. Leukemia. 2016). However, approaches for the specific therapeutic targeting of these patients are thus far lacking. 

Aims
Retroviral restoration of PU.1 expression has previously been explored but is difficult to achieve pharmacologically. Here, we tested the inverse strategy. As complete loss of PU.1 leads to stem cell failure, we hypothesized that AML cells harboring already low levels of PU.1 may be more vulnerable to further PU.1 inhibition. 

Methods

We used two alternative approaches: RNA interference and newly developed small molecule PU.1 inhibitors. 

Results

We found that inhibition of PU.1 with different shRNAs led to a significant decrease in proliferation and clonogenicity, and increased apoptosis of mouse and human leukemic cell lines with low PU.1 levels, as well as the majority of primary human AML cells tested.
The pharmacologic targeting of transcription factor-DNA major groove interactions is challenging. However, specific PU.1 binding to chromatin critically depends on additional minor groove contacts upstream of the core ETS binding motif, which determine selectivity for PU.1. We used an integrated screening strategy utilizing biosensor surface plasmon resonance, DNA footprinting, and cell-based dual-color PU.1 reporter assays to develop novel small molecules of the heterocyclic diamidine family as first-in-class PU.1 inhibitors. Targeted occupancy by our compounds in the minor groove induces perturbations in DNA conformation that are transmitted to the PU.1 site in the major groove and thus inhibit PU.1 binding via an allosteric mechanism. Functionally, treatment with 3 different compounds decreased cell growth and colony forming capacity, increased apoptosis, and disrupted serial replating capacity of PU.1low AML cell lines, and a majority of primary AML cell samples. ChIP and expression analysis showed that the compounds disrupt PU.1-promoter interaction and lead to downregulation of canonical PU.1 transcriptional targets in AML cells, confirming on-target activity. Genome-wide analysis showed highly significant enrichment of known transcriptional targets of PU.1, and selectivity over other ETS family members. Comparison with published transcriptomic and PU.1 ChIP-seq data sets, as well as ARACNe analysis of the PU.1 regulon in primary AML cells, demonstrated that the inhibitors antagonized PU.1-regulated pathways at a genome-wide level. Treatment of normal HSPC in colony forming assays led to decreased production of mature granulo-monocytic cells, consistent with PU.1’s known role in this lineage. However, this effect was reversible upon drug removal, and serial replating capacity was not affected suggesting no significant effects on normal HSPC. Lastly, in vivo treatment with PU.1 inhibitors in mouse and human AML (xeno)transplantation models significantly decreased tumor burden and increased survival.

Conclusion

Our study describes for the first time a strategy inhibiting PU.1 in AML, establishing proof-of-concept for this approach. Furthermore, we report the development of first-in-class PU.1 inhibitors which interfere with PU.1-DNA interaction through an allosteric, minor groove-mediated mechanism. Our work shows that it is feasible to pharmacologically target PU.1, and raises intriguing possibilities for the potential targeting of other transcription factors through minor groove-directed approaches.  

Session topic: 3. Acute myeloid leukemia - Biology

Keyword(s): transcription factor, Targeted therapy, PU.1, Acute Myeloid Leukemia

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