High-Dimensional Single-Cell Proteomic Analysis Reveals Therapeutic Vulnerabilities in Relapsed/Refractory AML By Concomitant Analysis of Cell Surface Antigens, Signaling Pathways and Anti-Apoptotic Proteins

• Published in: Blood 2020-11, Vol.136 (Supplement 1), p.26-26
• Main Authors: Muftuoglu, Muharrem, Alaniz, Zoe, Mak, Duncan, Lin, Angelique J., Burks, Jared K., Carter, Bing Z, Andreeff, Michael
• Format: Article
• Language: English
• ISSN: 0006-4971; 1528-0020
• DOI: 10.1182/blood-2020-143432

Background: The heterogeneous and adaptive nature of AML-associated genomic and proteomic landscape may account for disease relapse and poor prognosis, as therapy-associated selective pressure drives the emergence and expansion of AML clones with features different from those detected at diagnosis. The evolving focus is on single-cell analytical tools to fully capture the pathobiological heterogeneity of AML. We leveraged CyTOF to interrogate the proteomic heterogeneity in patients with R/R AML, which could potentially permit the design of rational combinatorial therapeutic approaches targeting vulnerabilities in these cells.. Methods: To dissect AML heterogeneity and its contribution to treatment failure, we designed a 51-parameter CyTOF panel and interrogated cellular hierarchy, major immune phenotypes, anti-apoptotic molecules, signaling pathways, exhaustion markers and attractive targets for CAR T-cell therapy in R/R AML (n:13). Results: First, we generated two-dimensional t-SNE maps and observed that the leukemic bone marrow compartment harbored immature (CD34+CD38- and CD34+CD38+) and mature leukemic blasts (CD33+CD34-) and major immune subsets. Constitutively active signaling pathways characterized by high levels of p-4EBP1, p-MEK1/2, p-S6 and p-AKT, marked immature and mature leukemia cells and comparative analysis revealed that monocytic blasts harbored more active signaling networks. The proportions of these subpopulations varied significantly across patients. We initially assessed the distribution of anti-apoptotic molecules across these leukemia compartments. Strikingly, Bcl-2 levels were considerably high within less-differentiated leukemic cell compartments and CD68 expressing leukemic blasts with monocytic differentiation had significantly lower levels of Bcl-2. This suggests that differentiated leukemic cells could preferentially survive under selection pressure of Bcl-2 inhibitors. On the contrary, we observed a trend towards higher Mcl-1 levels in differentiated leukemia cells. These findings provide a rationale for combining therapeutic modalities to target different leukemia subpopulations. Indeed, Bcl-2 and Mcl-1 inhibitors (Venetoclax and AZD5991) resulted in highly synergistic effects in AML PDX models. Hence, this analysis supports the hypothesis that Mcl-1 overexpression is a resistance factor to Bcl-2 inhibition, usually understood as developing in the same cell. Next, we assessed expression patterns of putative CAR T-cell targets