AML-354: High-Dimensional Single-Cell Proteomic Analysis Reveals Therapeutic Vulnerabilities in Patients with Relapsed/Refractory AML

Abstract

We designed a 51-parameter mass cytometry panel and interrogated acute myeloid leukemia (AML) heterogeneity to assess cellular hierarchy, anti-apoptotic molecules, exhaustion markers and targets for CAR T-cell therapy in R/R AML. We generated two-dimensional t-SNE maps to visualize distinct populations and observed significant heterogeneity among patients with regards to leukemic compartment composition. Notably, the leukemic bone marrow compartment harbored leukemia stem cells (CD34+CD38-), immature leukemic cells (CD34+CD38+), more differentiated leukemic blasts (CD33+CD34-) and major immune subsets. We initially assessed the distribution of anti-apoptotic molecules across leukemia compartments. Strikingly, Bcl-2 levels were considerably high within less-differentiated leukemic cell compartments and leukemic blasts with monocytic differentiation had significantly low levels of Bcl-2. On the contrary, we observed a trend towards higher Mcl-1 levels in differentiated leukemic cells. These findings provide a rationale for combining therapeutic modalities to target different leukemia subpopulations. Indeed, Bcl-2 and Mcl-1 inhibitors resulted in highly synergistic effects in AML PDX models (Carter et.al 2018, 2019). Of interest, Bcl-xl was ubiquitously expressed across leukemic and immune cell clusters. Next, we assessed expression patterns of CAR T-cell targets expressed on leukemic cells and identified significant variegated expression patterns for all CAR T-cell therapy targets in R/R AML samples. CD123 demonstrated patchy distribution across immature and differentiated leukemic blasts while CD33 expression was the main characteristic of differentiated leukemic blasts. Of note, the immature leukemia compartment demonstrated variable levels of CD33 and complete lack of CD33 on CD34+ leukemic cells was observed in a subgroup of patients. CLL-1 expression was the most promising marker and expressed across almost all leukemia compartments. Nevertheless, CLL-1 expression on leukemic cells was not ubiquitous in a small group of patients. In line with leukemic cell heterogeneity, we found a significant degree of variation in immune cell composition. Inhibitory molecules PD-1 and TIGIT were significantly expressed on CD4 and CD8 T-cells respectively. Single-cell profiling of R/R AML using mass cytometry reveals significant heterogeneous expression patterns of molecules targeted by CAR T-cells, immunotherapeutic approaches and targeted therapies. This approach provides a rationale to adopt combinatorial therapeutic approaches to target distinct leukemic populations with widely varying target expression.