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1.
Nat Cancer ; 3(7): 837-851, 2022 07.
Article in English | MEDLINE | ID: mdl-35668193

ABSTRACT

Selinexor is a first-in-class inhibitor of the nuclear exportin XPO1 that was recently approved by the US Food and Drug Administration for the treatment of multiple myeloma and diffuse large B-cell lymphoma. In relapsed/refractory acute myeloid leukemia (AML), selinexor has shown promising activity, suggesting that selinexor-based combination therapies may have clinical potential. Here, motivated by the hypothesis that selinexor's nuclear sequestration of diverse substrates imposes pleiotropic fitness effects on AML cells, we systematically catalog the pro- and anti-fitness consequences of selinexor treatment. We discover that selinexor activates PI3Kγ-dependent AKT signaling in AML by upregulating the purinergic receptor P2RY2. Inhibiting this axis potentiates the anti-leukemic effects of selinexor in AML cell lines, patient-derived primary cultures and multiple mouse models of AML. In a syngeneic, MLL-AF9-driven mouse model of AML, treatment with selinexor and ipatasertib outperforms both standard-of-care chemotherapy and chemotherapy with selinexor. Together, these findings establish drug-induced P2RY2-AKT signaling as an actionable consequence of XPO1 inhibition in AML.


Subject(s)
Leukemia, Myeloid, Acute , Proto-Oncogene Proteins c-akt , Animals , Antineoplastic Combined Chemotherapy Protocols , Karyopherins/antagonists & inhibitors , Leukemia, Myeloid, Acute/drug therapy , Mice , Proto-Oncogene Proteins c-akt/metabolism , Receptors, Cytoplasmic and Nuclear/antagonists & inhibitors , Receptors, Cytoplasmic and Nuclear/metabolism , Receptors, Purinergic P2Y2/metabolism , United States , Exportin 1 Protein
2.
Blood Cancer J ; 12(6): 95, 2022 06 24.
Article in English | MEDLINE | ID: mdl-35750691

ABSTRACT

Functional precision medicine in AML often relies on short-term in vitro drug sensitivity screening (DSS) of primary patient cells in standard culture conditions. We designed a niche-like DSS assay combining physiologic hypoxia (O2 3%) and mesenchymal stromal cell (MSC) co-culture with multiparameter flow cytometry to enumerate lymphocytes and differentiating (CD11/CD14/CD15+) or leukemic stem cell (LSC)-enriched (GPR56+) cells within the leukemic bulk. After functional validation of GPR56 expression as a surrogate for LSC enrichment, the assay identified three patterns of response, including cytotoxicity on blasts sparing LSCs, induction of differentiation, and selective impairment of LSCs. We refined our niche-like culture by including plasma-like amino-acid and cytokine concentrations identified by targeted metabolomics and proteomics of primary AML bone marrow plasma samples. Systematic interrogation revealed distinct contributions of each niche-like component to leukemic outgrowth and drug response. Short-term niche-like culture preserved clonal architecture and transcriptional states of primary leukemic cells. In a cohort of 45 AML samples enriched for NPM1c AML, the niche-like multiparametric assay could predict morphologically (p = 0.02) and molecular (NPM1c MRD, p = 0.04) response to anthracycline-cytarabine induction chemotherapy. In this cohort, a 23-drug screen nominated ruxolitinib as a sensitizer to anthracycline-cytarabine. This finding was validated in an NPM1c PDX model.


Subject(s)
Leukemia, Myeloid, Acute , Mesenchymal Stem Cells , Anthracyclines/metabolism , Anthracyclines/therapeutic use , Cytarabine/therapeutic use , Drug Evaluation, Preclinical , Humans , Leukemia, Myeloid, Acute/diagnosis , Leukemia, Myeloid, Acute/drug therapy , Leukemia, Myeloid, Acute/genetics , Mesenchymal Stem Cells/metabolism , Neoplastic Stem Cells/metabolism
3.
Leukemia ; 36(6): 1585-1595, 2022 06.
Article in English | MEDLINE | ID: mdl-35474100

ABSTRACT

By querying metabolic pathways associated with leukemic stemness and survival in multiple AML datasets, we nominated SLC7A11 encoding the xCT cystine importer as a putative AML dependency. Genetic and chemical inhibition of SLC7A11 impaired the viability and clonogenic capacity of AML cell lines in a cysteine-dependent manner. Sulfasalazine, a broadly available drug with xCT inhibitory activity, had anti-leukemic activity against primary AML samples in ex vivo cultures. Multiple metabolic pathways were impacted upon xCT inhibition, resulting in depletion of glutathione pools in leukemic cells and oxidative stress-dependent cell death, only in part through ferroptosis. Higher expression of cysteine metabolism genes and greater cystine dependency was noted in NPM1-mutated AMLs. Among eight anti-leukemic drugs, the anthracycline daunorubicin was identified as the top synergistic agent in combination with sulfasalazine in vitro. Addition of sulfasalazine at a clinically relevant concentration significantly augmented the anti-leukemic activity of a daunorubicin-cytarabine combination in a panel of 45 primary samples enriched in NPM1-mutated AML. These results were confirmed in vivo in a patient-derived xenograft model. Collectively, our results nominate cystine import as a druggable target in AML and raise the possibility to repurpose sulfasalazine for the treatment of AML, notably in combination with chemotherapy.


Subject(s)
Cystine , Leukemia, Myeloid, Acute , Cell Line, Tumor , Cysteine , Cystine/metabolism , Cystine/therapeutic use , Daunorubicin/pharmacology , Daunorubicin/therapeutic use , Humans , Leukemia, Myeloid, Acute/drug therapy , Leukemia, Myeloid, Acute/genetics , Nuclear Proteins , Sulfasalazine/pharmacology , Sulfasalazine/therapeutic use
4.
Sci Transl Med ; 13(587)2021 03 31.
Article in English | MEDLINE | ID: mdl-33790022

ABSTRACT

The development and survival of cancer cells require adaptive mechanisms to stress. Such adaptations can confer intrinsic vulnerabilities, enabling the selective targeting of cancer cells. Through a pooled in vivo short hairpin RNA (shRNA) screen, we identified the adenosine triphosphatase associated with diverse cellular activities (AAA-ATPase) valosin-containing protein (VCP) as a top stress-related vulnerability in acute myeloid leukemia (AML). We established that AML was the most responsive disease to chemical inhibition of VCP across a panel of 16 cancer types. The sensitivity to VCP inhibition of human AML cell lines, primary patient samples, and syngeneic and xenograft mouse models of AML was validated using VCP-directed shRNAs, overexpression of a dominant-negative VCP mutant, and chemical inhibition. By combining mass spectrometry-based analysis of the VCP interactome and phospho-signaling studies, we determined that VCP is important for ataxia telangiectasia mutated (ATM) kinase activation and subsequent DNA repair through homologous recombination in AML. A second-generation VCP inhibitor, CB-5339, was then developed and characterized. Efficacy and safety of CB-5339 were validated in multiple AML models, including syngeneic and patient-derived xenograft murine models. We further demonstrated that combining DNA-damaging agents, such as anthracyclines, with CB-5339 treatment synergizes to impair leukemic growth in an MLL-AF9-driven AML murine model. These studies support the clinical testing of CB-5339 as a single agent or in combination with standard-of-care DNA-damaging chemotherapy for the treatment of AML.


Subject(s)
Antineoplastic Agents , Leukemia, Myeloid, Acute , Adenosine Triphosphatases/metabolism , Animals , Antineoplastic Agents/therapeutic use , Cell Line, Tumor , DNA Repair , Humans , Leukemia, Myeloid, Acute/drug therapy , Mice , Valosin Containing Protein
5.
Cancer Discov ; 10(12): 1894-1911, 2020 12.
Article in English | MEDLINE | ID: mdl-32826232

ABSTRACT

Deciphering the impact of metabolic intervention on response to anticancer therapy may elucidate a path toward improved clinical responses. Here, we identify amino acid-related pathways connected to the folate cycle whose activation predicts sensitivity to MYC-targeting therapies in acute myeloid leukemia (AML). We establish that folate restriction and deficiency of the rate-limiting folate cycle enzyme MTHFR, which exhibits reduced-function polymorphisms in about 10% of Caucasians, induce resistance to MYC targeting by BET and CDK7 inhibitors in cell lines, primary patient samples, and syngeneic mouse models of AML. Furthermore, this effect is abrogated by supplementation with the MTHFR enzymatic product CH3-THF. Mechanistically, folate cycle disturbance reduces H3K27/K9 histone methylation and activates a SPI1 transcriptional program counteracting the effect of BET inhibition. Our data provide a rationale for screening MTHFR polymorphisms and folate cycle status to nominate patients most likely to benefit from MYC-targeting therapies. SIGNIFICANCE: Although MYC-targeting therapies represent a promising strategy for cancer treatment, evidence of predictors of sensitivity to these agents is limited. We pinpoint that folate cycle disturbance and frequent polymorphisms associated with reduced MTHFR activity promote resistance to BET inhibitors. CH3-THF supplementation thus represents a low-risk intervention to enhance their effects.See related commentary by Marando and Huntly, p. 1791.This article is highlighted in the In This Issue feature, p. 1775.


Subject(s)
Folic Acid/metabolism , Methylenetetrahydrofolate Reductase (NADPH2)/metabolism , Neoplasms/drug therapy , Neoplasms/metabolism , Animals , Clustered Regularly Interspaced Short Palindromic Repeats , Drug Resistance, Neoplasm , Humans , Male , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , Mice, Knockout , Molecular Targeted Therapy , Proto-Oncogene Proteins c-myc/biosynthesis , U937 Cells
6.
Blood ; 135(14): 1111-1123, 2020 04 02.
Article in English | MEDLINE | ID: mdl-32027737

ABSTRACT

Hematopoietic stem cell (HSC) transplantation (HSCT) is often exploited to treat hematologic disease. Donor HSCs must survive, proliferate, and differentiate in the damaged environment of the reconstituting niche. Illuminating molecular mechanisms regulating the activity of transplanted HSCs will inform efforts to improve HSCT. Here, we report that G-protein-coupled receptor-associated sorting proteins (GPRASPs) function as negative regulators of HSCT. Silencing of Gprasp1 or Gprasp2 increased the survival, quiescence, migration, niche retention, and hematopoietic repopulating activity of hematopoietic stem and progenitor cells (HSPCs) posttransplant. We further show that GPRASP1 and GPRASP2 promote the degradation of CXCR4, a master regulator of HSC function during transplantation. CXCR4 accumulates in Gprasp-deficient HSPCs, boosting their function posttransplant. Thus, GPRASPs negatively regulate CXCR4 stability in HSCs. Our work reveals GPRASP proteins as negative regulators of HSCT and CXCR4 activity. Disruption of GPRASP/CXCR4 interactions could be exploited in the future to enhance the efficiency of HSCT.


Subject(s)
Hematopoietic Stem Cell Transplantation , Hematopoietic Stem Cells/cytology , Intracellular Signaling Peptides and Proteins/metabolism , Animals , Carrier Proteins , Cell Movement , Cell Proliferation , Cell Survival , Gene Deletion , Gene Silencing , Hematopoietic Stem Cell Transplantation/methods , Hematopoietic Stem Cells/metabolism , Humans , Intracellular Signaling Peptides and Proteins/genetics , Mice , Mice, Inbred C57BL , Proteolysis , Receptors, CXCR4/metabolism , Receptors, G-Protein-Coupled/genetics , Receptors, G-Protein-Coupled/metabolism
7.
Mol Cell ; 72(6): 955-969.e7, 2018 12 20.
Article in English | MEDLINE | ID: mdl-30576657

ABSTRACT

The fidelity of transcription initiation is essential for accurate gene expression, but the determinants of start site selection are not fully understood. Rap1 and other general regulatory factors (GRFs) control the expression of many genes in yeast. We show that depletion of these factors induces widespread ectopic transcription initiation within promoters. This generates many novel non-coding RNAs and transcript isoforms with diverse stability, drastically altering the coding potential of the transcriptome. Ectopic transcription initiation strongly correlates with altered nucleosome positioning. We provide evidence that Rap1 can suppress ectopic initiation by a "place-holder" mechanism whereby it physically occludes inappropriate sites for pre-initiation complex formation. These results reveal an essential role for GRFs in the fidelity of transcription initiation and in the suppression of pervasive transcription, profoundly redefining current models for their function. They have important implications for the mechanism of transcription initiation and the control of gene expression.


Subject(s)
Gene Expression Regulation, Fungal , RNA, Fungal/biosynthesis , RNA, Messenger/biosynthesis , RNA, Untranslated/metabolism , Saccharomyces cerevisiae Proteins/metabolism , Saccharomyces cerevisiae/metabolism , Telomere-Binding Proteins/metabolism , Transcription Factors/metabolism , Transcription, Genetic , Binding Sites , Chromatin Assembly and Disassembly , Nucleosomes/genetics , Nucleosomes/metabolism , Promoter Regions, Genetic , Protein Binding , RNA, Fungal/genetics , RNA, Messenger/genetics , RNA, Untranslated/genetics , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae Proteins/genetics , Shelterin Complex , Telomere-Binding Proteins/genetics , Transcription Factors/genetics , Transcription Initiation Site , Transcription Initiation, Genetic
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