Type: Oral
Session: 802. Chemical Biology and Experimental Therapeutics: Innovations in Therapy and Drug Screening
Hematology Disease Topics & Pathways:
AML, HSCs, Adult, Diseases, Elderly, cellular interactions, Biological Processes, Technology and Procedures, Cell Lineage, Study Population, Myeloid Malignancies, flow cytometry, metabolomics, microenvironment
Objectives. To develop an ex vivo niche-like multiparametric DSS platform for primary AML cells. To validate its clinical relevance in NPM1mut pts treated with conventional DNR-AraC chemotherapy. To discover new sensitizers to DNR-AraC chemotherapy in NPM1mut AML.
Results. We designed an MFC panel to count viable blasts and measure their differentiation (CD11b/CD14/CD15) and stemness (GPR56) after exclusion of residual lymphocytes (Figure 1A). We validated GPR56 expression as stemness marker based on increased retention of GPR56+ cells in niche-like coculture combining hypoxia (O2 3%) and MSC compared to standard conditions (p<0.0001, Figure 1B) and limit dilution assays of residual GPR56+ cells at 72h of niche-like culture in 3 NPM1mut AMLs (Figure 1C). Using a limited panel of 14 drugs or combinations at fixed concentrations, our MFC readout after 72h of coculture with MSC+hypoxia revealed the distinct mode of action of different agents or combinations including the differentiation activity of ATO-ATRA, the LSC-sparring cytotoxicity of DNR-AraC and the anti-LSC- activity of VEN (Figure 1D).
To further mimic in vivo conditions, we derived a MEMa-based plasma-like medium (PLM) based on targeted metabolomics (Figure 1E) and electro-chemoluminescent cytokine assays of 29 diagnostic AML bone marrow plasma samples compared to conditioned media of primary AML cells cultured in niche-like conditions (MSC, hypoxia). This instructed the design of our custom PLM with dialyzed FBS and defined low-dose (~1 ng/mL range) cytokines (CK) and amino-acid (AA) concentrations.
We next investigated the contribution of MSCs, hypoxia, plasma-like AAs and CKs on blasts viability, differentiation, stemness and drug response in 3 NPM1mut AMLs exposed to fixed concentrations of 6 core AML therapies. This analysis uncovered significant interactions between these 4 niche components in dictating blast viability and stemness upon 72h ex vivo culture (Figure 1F) and revealed the distinct contribution of these niche components to drug sensitivity. RNA-seq of primary blasts cultured in niche-like, plasma-like conditions revealed marked enrichment of stemness pathways compared to ex vivo culture in standard conditions.
Finally, we explored DNR-AraC (five-point serial dilution) alone or in combination with fixed, clinically relevant concentrations of 24 drugs in 49 primary AML samples (including 34 NPM1mut). Using AUCs of DNR-AraC on lymphocytes as internal control, we first validated our NEXT assay on NPM1 MRD levels in the 34 NPM1mut pts treated frontline with conventional DNR-AraC regimens (Figure 1G). Across all 49 pts, we uncovered 11 different optimal ‘third-drugs’, stressing the role of our NEXT assay to deploy precision medicine in daily practice. At the population level, we could nominate 3 top combinations, two of which are currently in clinical investigation (Venetoclax and Selinexor). The unpublished sensitizing effect of low dose (0.25µM) Ruxolitinib on DNR-AraC uncovered with our NEXT assay is currently being investigated in PDX models.
Conclusion. We designed the NEXT assay, a multiparametric drug screening of AML viability, differentiation and stemness in niche-like culture combining hypoxia, stromal interactions and plasma-like medium. Components of the niche-like culture interact to govern leukemic viability and stemness. Our assay could predict MRD achievement in NPM1mut AML and identifies novel sensitizers to DNR-AraC in these pts.
Disclosures: Clappier: Amgen: Honoraria, Research Funding. Ades: Abbvie: Honoraria, Membership on an entity's Board of Directors or advisory committees; takeda: Honoraria, Membership on an entity's Board of Directors or advisory committees; jazz: Membership on an entity's Board of Directors or advisory committees, Research Funding; Amgen: Research Funding; novartis: Research Funding; Celgene/BMS: Research Funding. Itzykson: Amgen: Membership on an entity's Board of Directors or advisory committees; Otsuka Pharma: Membership on an entity's Board of Directors or advisory committees; Jazz Pharmaceuticals: Honoraria, Membership on an entity's Board of Directors or advisory committees; Stemline: Membership on an entity's Board of Directors or advisory committees; Oncoethix (now Merck): Research Funding; Janssen: Research Funding; Karyopharm: Membership on an entity's Board of Directors or advisory committees; Abbvie: Honoraria; Daiichi Sankyo: Honoraria; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; BMS (Celgene): Honoraria; Sanofi: Honoraria; Astellas: Honoraria.