Leukemia Cell of Origin Influences p53 Activity and Therapeutic Sensitivity Via an Evi1-Dependent Mechanism
Authors: Cai Sheng F , Chu S Haihua , Goldberg Aaron D , Parvin Salma , Koche Richard , Glass Jacob L , Stein Eytan M. , Tallman Martin S. , Sen Filiz , Famulare Christopher , Cusan Monica , Huang Chun-Hao , Chen Chun-Wei David , Zou Lihua , Cordner Keith B , Durani Vidushi , Kini Mitali , Rex Madison , Zuber Johannes , Baslan Timour , Lowe Scott W. , Rienhoff Hugh Young , Levine Ross L. , Armstrong Scott A. 
Published: February 2020

Prior studies have shown that the cell of origin of acute myeloid leukemia (AML) initiation is an important determinant of therapeutic sensitivity. MLL-rearranged leukemias in which the fusion is acquired in hematopoietic stem cells (HSC) are less sensitive to chemotherapy and express high levels of the oncogenic transcription factor, Evi1, when compared to leukemias which are initiated in granulocyte-macrophage progenitors (GMP). However, the mechanisms governing how cell-of-origin modulates therapeutic response have not been delineated. Here, we describe a functional link that ties therapeutic sensitivity of MLL-AF9 leukemias to both chemotherapy and pharmacologic inhibitors of lysine-specific demethylase 1 (LSD1) - currently under investigation in clinical trials - to AML cell of origin via a novel mechanism that modulates p53 protein stability and activity through Evi1. This Evi1-dependent mechanism revealed a therapeutic vulnerability in the resistant HSC-derived leukemias that could be targeted with the BCL2 inhibitor venetoclax, which overcame resistance when combined with LSD1 inhibition.
Murine HSC-derived MLL-AF9 leukemias exhibited markedly reduced sensitivity to the LSD1 inhibitor, IMG-7289, when compared to GMP-derived MLL-AF9 leukemias, in vitro and in vivo. Consistent with previously published reports, HSC-derived leukemias exhibited several hundred-fold higher expression of EVI1 mRNA relative to GMP-derived leukemias in both mouse and human MLL-rearranged leukemia models; the differential EVI1 expression mirrors expression patterns of this key hematopoietic regulator in normal HSCs and GMPs. H3K27me3 chromatin immunoprecipitation coupled with high-throughput sequencing (ChIP-seq) revealed that the EVI1 locus is silenced by Polycomb repression in GMP-derived leukemias, and this repression was also observed in EVI1low primary AML patient samples. Knockdown of Evi1 via shRNA sensitized HSC-derived leukemias to LSD1 inhibition.
In vitro assays revealed induction of apoptosis in GMP-derived leukemias - but not in HSC-derived leukemias - after treatment with IMG-7289. HSC-derived leukemias also exhibited decreased apoptotic priming assessed through functional BH3 profiling assays as well as blunted p53 transcriptional output. These data suggested that cell-of-origin led to differential p53 activity and therapeutic response in AMLs driven by the same fusion gene initiated from different stem/progenitor populations. The diminished p53 activity in HSC-derived leukemias was associated with reduced p53 protein abundance both at steady-state and after LSD1 inhibitor treatment compared to GMP-derived leukemias. Quantification of p53 mRNA did not show differential p53 expression, including after LSD1 inhibition, in HSC vs. GMP-derived leukemias implicating post-transcriptional regulatory mechanisms that underlie this cell-of-origin mediated phenotype. We found that modulation of Evi1 expression resulted in altered p53 protein stability: specifically, (1) shRNA-mediated knockdown of Evi1 in HSC-derived leukemias increased p53 protein stability and (2) overexpression of Evi1 blunted doxorubicin-induced p53 protein stability. Moreover, p53 loss-of-function in Evi1low GMP-derived MLL-AF9 leukemias induced resistance to LSD1 inhibition. By contrast, Evi1high HSC-derived leukemias exposed to the BCL2 inhibitor venetoclax in vivo were sensitized to LSD1 inhibition, resulting in enhanced apoptosis and greater reductions in disease burden, observations that we observed in patients with Evi1high AML treated with venetoclax. Our findings describe how the cell of origin of p53 wild-type cancers can differentially modulate p53 function and therapeutic response and provide a mechanistic rationale for therapies aimed to circumvent this resistance mechanism.

Disclosures
Cai: Imago Biosciences, Inc.: Consultancy. Goldberg:Celgene: Consultancy; Daiichi-Sankyo: Consultancy, Research Funding; Pfizer: Research Funding; Arog Pharmaceuticals: Research Funding; ADC Therapeutics: Research Funding; American Society of Clinical Oncology: Research Funding; Abbvie: Consultancy; Abbvie: Research Funding; American Society of Hematology: Research Funding; DAVA Oncology: Honoraria. Stein:Agios: Consultancy, Membership on an entity's Board of Directors or advisory committees; Astellas Pharma US, Inc: Membership on an entity's Board of Directors or advisory committees; Celgene Corporation: Membership on an entity's Board of Directors or advisory committees; Daiichi Sankyo, Inc.: Membership on an entity's Board of Directors or advisory committees; Bioline: Membership on an entity's Board of Directors or advisory committees; Genentech: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees; PTC Therapeutics: Membership on an entity's Board of Directors or advisory committees; Syros: Membership on an entity's Board of Directors or advisory committees. Tallman:Daiichi-Sankyo: Consultancy, Membership on an entity's Board of Directors or advisory committees; Abbvie: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Jazz Pharmaceuticals: Consultancy, Membership on an entity's Board of Directors or advisory committees; BioLineRx: Consultancy, Membership on an entity's Board of Directors or advisory committees; Oncolyze: Consultancy, Membership on an entity's Board of Directors or advisory committees; Cellerant: Research Funding; ADC Therapeutics: Research Funding; BioLineRx: Consultancy, Membership on an entity's Board of Directors or advisory committees; BioLineRx: Consultancy, Membership on an entity's Board of Directors or advisory committees; Daiichi-Sankyo: Consultancy, Membership on an entity's Board of Directors or advisory committees; KAHR: Consultancy, Membership on an entity's Board of Directors or advisory committees; KAHR: Consultancy, Membership on an entity's Board of Directors or advisory committees; Delta Fly Pharma: Consultancy, Membership on an entity's Board of Directors or advisory committees; Delta Fly Pharma: Consultancy, Membership on an entity's Board of Directors or advisory committees; Rigel: Consultancy, Membership on an entity's Board of Directors or advisory committees; Nohla: Consultancy, Membership on an entity's Board of Directors or advisory committees; Tetraphase: Consultancy, Membership on an entity's Board of Directors or advisory committees; Tetraphase: Consultancy, Membership on an entity's Board of Directors or advisory committees; Nohla: Consultancy, Membership on an entity's Board of Directors or advisory committees; Delta Fly Pharma: Consultancy, Membership on an entity's Board of Directors or advisory committees; UpToDate: Patents & Royalties; UpToDate: Patents & Royalties; Abbvie: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Oncolyze: Consultancy, Membership on an entity's Board of Directors or advisory committees; Jazz Pharmaceuticals: Consultancy, Membership on an entity's Board of Directors or advisory committees; ADC Therapeutics: Research Funding; Cellerant: Research Funding; Abbvie: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; UpToDate: Patents & Royalties; Cellerant: Research Funding; Daiichi-Sankyo: Consultancy, Membership on an entity's Board of Directors or advisory committees; BioLineRx: Consultancy, Membership on an entity's Board of Directors or advisory committees; Orsenix: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Cellerant: Research Funding; Biosight: Research Funding; Biosight: Research Funding; ADC Therapeutics: Research Funding; ADC Therapeutics: Research Funding; Orsenix: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; ADC Therapeutics: Research Funding; Cellerant: Research Funding; Abbvie: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; 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Tetraphase: Consultancy, Membership on an entity's Board of Directors or advisory committees; Oncolyze: Consultancy, Membership on an entity's Board of Directors or advisory committees; Oncolyze: Consultancy, Membership on an entity's Board of Directors or advisory committees; Jazz Pharmaceuticals: Consultancy, Membership on an entity's Board of Directors or advisory committees; Tetraphase: Consultancy, Membership on an entity's Board of Directors or advisory committees; Jazz Pharmaceuticals: Consultancy, Membership on an entity's Board of Directors or advisory committees; Jazz Pharmaceuticals: Consultancy, Membership on an entity's Board of Directors or advisory committees; Jazz Pharmaceuticals: Consultancy, Membership on an entity's Board of Directors or advisory committees; Orsenix: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Abbvie: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Daiichi-Sankyo: Consultancy, Membership on an entity's Board of Directors or advisory committees; UpToDate: Patents & Royalties; Delta Fly Pharma: Consultancy, Membership on an entity's Board of Directors or advisory committees; Abbvie: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Delta Fly Pharma: Consultancy, Membership on an entity's Board of Directors or advisory committees; Rigel: Consultancy, Membership on an entity's Board of Directors or advisory committees; Daiichi-Sankyo: Consultancy, Membership on an entity's Board of Directors or advisory committees; KAHR: Consultancy, Membership on an entity's Board of Directors or advisory committees; Rigel: Consultancy, Membership on an entity's Board of Directors or advisory committees; Rigel: Consultancy, Membership on an entity's Board of Directors or advisory committees; Daiichi-Sankyo: Consultancy, Membership on an entity's Board of Directors or advisory committees; Nohla: Consultancy, Membership on an entity's Board of Directors or advisory committees; 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Lowe:ORIC pharmaceuticals: Consultancy, Equity Ownership; Mirimus: Consultancy, Equity Ownership; Constellation Pharma: Consultancy, Equity Ownership; Petra Pharmaceuticals: Consultancy, Equity Ownership; PMV Pharmaceuticals: Consultancy, Equity Ownership; Faeth Therapeutics: Consultancy, Equity Ownership; Blueprint Medicines: Consultancy, Equity Ownership. Rienhoff:Imago Biosciences: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties. Levine:Loxo: Membership on an entity's Board of Directors or advisory committees; Lilly: Honoraria; Amgen: Honoraria; Celgene: Consultancy, Research Funding; Imago Biosciences: Membership on an entity's Board of Directors or advisory committees; Prelude Therapeutics: Research Funding; Qiagen: Membership on an entity's Board of Directors or advisory committees; Gilead: Consultancy; Isoplexis: Membership on an entity's Board of Directors or advisory committees; Novartis: Consultancy; Roche: Consultancy, Research Funding; C4 Therapeutics: Membership on an entity's Board of Directors or advisory committees. 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