ASXL1 Truncating Mutations Drive Leukemic Resistance to T Cell Attack

Restoration of graft-versus leukemia activity underlies the efficacy of donor lymphocyte infusion (DLI), a curative immunotherapy for leukemic relapse following allogeneic stem cell transplant (SCT). We found that while specific exhausted T cell subsets defined DLI response, no pattern of T cell dysfunction appeared in nonresponders (NRs) [PMID: 34758319]. We hypothesized that leukemic molecular pathways define DLI resistance (DLIr).

We performed whole exome sequencing on bone marrow samples collected before and after DLI from 11 patients (pts) with relapsed chronic myeloid leukemia (CML) at Dana-Farber Cancer Institute. We used MuTect2 to discover somatic alterations using genomic DNA from recipient-derived mesenchymal stromal cells and donor peripheral blood cells as germline sources. The burden of nonsynonymous mutations and mutational spectra was similar between responders (Rs) and non-responders (NRs). Known non-Philadelphia (Ph) chromosome cancer drivers were found in 0/7 Rs and 4/4 NRs (RUNX1 (1/4), IKZF1 (2/4), and truncating exon 12 ASXL1 mutations (ASXL1 ^m) (3/4)). We profiled 6 more DLI-treated pts with relapsed CML from the MD Anderson Cancer Center with a targeted mutation panel that identified ASXL1 ^m in 4/5 NRs. Altogether, we found ASXL1 ^m in 78% (7/9) of NRs and 0% (0/10) of Rs (p=7e-4, two-sided Fisher's test), suggesting ASXL1 ^m as a driver of DLIr.

To discover molecular pathways of DLIr, we interrogated unpublished non T-cell single cell (sc)RNA-seq data in our DLI cohort (8 R, 6 NR). Leukemic cells were identified for patients (n=8) with sex-mismatched SCT by male sex-defining genes, which correlated with Ph chromosomal positivity (Pearson correlation = 0.85). These cells were primarily confined to the myeloid population, so we performed a scRNA myeloid reclustering (138,152 cells) with Harmony batch correction and identified 5 populations enriched in R vs NR; 4 clusters in NR, and 1 monocyte cluster in R (p=0.039, 0.005, 0.038, 0.043, and 0.025, respectively). These NR clusters were enriched in progenitor cell identities, notably the LSC17 [PMID: 27926740] signature (p<2e-16). Interrogation of immune pathways in the LSC cluster showed downregulation of HLA-I and multiple antigen processing/presentation (APP) genes in NRs (p<2e-16), while HLA-II remained unchanged. Thus, ASXL1 ^m may mediate immune escape through suppression of HLA-I expression and antigen presentation.

To directly test this hypothesis, we CRISPR corrected the endogenous ASXL1 ^m (Y591*) in a BCR-ABL-driven leukemic cell line (K562). This wildtype reversion (ASXL1 ^c) increased HLA-I expression at the RNA (RNA-seq; HLA-B/C, log2foldchange= -4.77, q=7e-42) and protein level (flow cytometry; 49.0% vs 1.62%, q=2e-4); here too, HLA-II expression remained unaffected by ASXL1 mutation status. Gene-set enrichment analysis also showed increased APP expression in ASXL1 ^c K562 (NES=-1.75, q=0.01).

We then sought to identify whether correction of ASXL1m acted through known regulators of HLA-I. Treatment with IFNɣ or EPZ011989, an inhibitor of enhancer of zeste-homolog 2 (EZH2), resulted in greater HLA-I in K562 ASXL1 ^c (85.0% vs 50.2% and 89.5% vs 39.3%, respectively, q<0.001) suggesting that ASXL1 ^cacts orthogonally to IFNɣ or EZH2. ASXL1 ^c also mediated increased T-cell cytotoxicity towards K562 ASXL1 ^c vs ASXL1 ^m without (43% vs 16%, q=2e-4) and with IFNɣ pre-treatment (71% vs 34%, q=1e-6) at 10:1 target to effector ratios. Finally, to study other disease contexts, we examined published sc chromatin accessibility and scRNA-seq data from a chronic myelomonocytic leukemia cohort [PMID: 35301312] and an acute myeloid leukemia cohort [PMID:32649887], respectively. We found both chromatin and transcriptional states enriched in ASXL1m samples to have decreased accessibility and expression, respectively, of HLA-I and APP genes (p<2E-16).

In summary, we integrated molecular analyses with immuno-functional studies to define a novel oncogenetic pathway of immune evasion. Specifically, we link DLIr to ASXL1 ^m that suppresses transcription of HLA-I and antigen presentation machinery in myeloid diseases; moreover, its correction restores anti-leukemic CD8+ T-cell cytotoxicity. Ongoing studies will examine additional clinical contexts and the epigenetic mechanisms by which ASXL1m regulates HLA-I expression.