Article Text
PDF
Abstract
Background Allogeneic stem cell transplantation can cure relapsed/refractor (R/R) AML via grafted T cells versus leukemia effect, but not a viable option to many patients. By combining azacitidine with nivolumab, we harnessed T cell activity and demonstrated 33% response rates. The tumor microenvironment (TME) factors impacting response and resistance to PD-1 blockade-based treatment in AML are unknown.
Methods We performed single cell RNA sequencing (scRNAseq) on 113,394 bone marrow (BM) cells, paired with >30,000 single cell T cell receptor (scTCR) repertoires, from 8 pre- and 14 post- azacitidine/nivolumab treatment aspirates of 8 R/R AML patients (median age 73 years; 3 responders; 3 non-responders; 2 stable disease) (figure 1).
Results Inferred copy number loss of chromosome 7/7q (chr7/7q) by scRNAseq was associated with resistance to azacitidine/nivolumab (figure 2A), which was validated in a larger cohort based on clinical karyotyping (figure 2B). There was significant enrichment (q<0.005) for IFNg pathway in chr7/7q. We identified marked variation in the T cell components across AML patients at pre- and post- treatment, demonstrating significant dynamic changes in CD4, CD8 and non-classical T cells populations, including MAIT (figure 3A-B). Among CD8 cells, we identified a unique GZMK-enriched population that was highest at pretreatment in responders. Pseudotemporal trajectory analysis revealed a continuum of CD8 cell states, intermediated by the less exhausted, GZMK-enriched CD8 population (figure 3C-D). GZMK also discriminated between 2 MAIT populations. GZMK-enriched cells had increased expression of the stem-like T cell transcription factor TCF7, and the T cell memory transcription factor EOMES. GZMK expression was associated with improved survival in de novo TCGA AML cohort (p=0.0017). scTCR clonotype assessment revealed shared clonotypes with the terminally effector CD8 CTL cells following PD-1 blockade. Following treatment, novel clones represented 38.7% (39/101) of total clones, followed by contracted clones (32.6%) and expanded (28.7%) clones (figure 3E-F). However, 76.9% and 72.4% of novel and expanded clones were contributed by the responders. Non-responders contributed only 5% and 3.4% of the novel and expanded clones, respectively.
Clinical design summarizing the age, response type per ELN, time to response, treatment frequency and the number of cells analyzed per patient
(A) Inferred copy number variation of representative 300 cells per patient at pretreatment (B) Correlation analysis for azacitidine/nivolumab based on chromosome 7/7q deletion
(A) UMAP of T cell subsets and (B) distribution of T cell subsets at different treatment timepoints(C-D) Monocle3-based pseudotemporal analysis of CD8 subsets(E) Scatterplots of clonotypes change of pre- versus post-treatment and by response groups(F) Number of novel, expanded and contracted clonotypes by response group
Conclusions Chr7/7q loss is associated with resistance to PD-1 blockade. CD8 cells exist in a continuum in BMs of patients with AML and GZMK expression identifies a stem-like, memory T cell subset. The subverted T cells can be reinvigorated via PD-1 blockade and induce responses in AML driven via novel and expanded clones demonstrating AML T cell plasticity and adaptability. Further functional characterization of GZMK expressing lymphocytes in mediating antileukemic responses is underway.
Ethics Approval The study was approved by IRB at MD Anderson Cancer Center
This is an open access article distributed in accordance with the Creative Commons Attribution 4.0 Unported (CC BY 4.0) license, which permits others to copy, redistribute, remix, transform and build upon this work for any purpose, provided the original work is properly cited, a link to the licence is given, and indication of whether changes were made. See: https://creativecommons.org/licenses/by/4.0/.