Identifying epigenetic vulnerabilities for treatment of HMA refractory myeloid leukemia Free

Epigenetic mutations (e.g. TETE2, ASXL1, DNMT3A) are a frequently occurring class of mutations in Acute Myeloid Leukemias (AMLs). One of the commonly used therapies in AML is hypomethylating agent (HMA) with venetoclax. Despite complete remissions as high as 60%, it is not a curative regimen with resistance and relapse developing in nearly all patients. HMAs act as epigenetic agents demethylating DNA but how this contributes to synergy with venetoclax is not well understood. We hypothesize HMAs induce an epigenetic-mediated differentiation effect that promotes venetoclax efficacy. Additional epigenetic agents and cell death inducers may be modeled after the HMA/Ven combination and have therapeutic potential in cases of HMA failure. We have previously developed a Tet2 Flt3 – mutant model of leukemia that demonstrates in vivo sensitivity to HMA and utilize it to study HMA resistance and additional epigenetic vulnerabilities.

Methods To understand HMA's activity, we developed a resistance model of leukemia through repeated treatment cycles. We characterized these cells through RNA-seq and ATAC-seq. We also screened leukemia cell lines and primary mouse cells for potential epigenetic targets using CRISPR. We then tested in vitro anti-leukemic activity of epigenetic agents.

Results We treated a Tet2 Flt3 leukemia mouse model through several rounds of HMA therapy and identified a resistant clone. In HMA-sensitive models platelets increased two weeks after HMA therapy (mean, 441 k/µl to 781 k/µl) whereas in the resistant clone platelet counts were lower to start and recovered less (mean, 180 k/µl to 269 k/µl). Similarly, more significant anemia and lower recovery (Sens, 46% to 41%; Res, 34% to 17%), but the white blood cell count still responded and declined following HMA (Sens, 18 k/µl to 8.4 k/µl; Res, 43 k/µl to 11.4 k/µl). Resistant cells had altered gene-expression in stem-progenitor cells by RNA-seq. Using GSEA analysis, we identified signatures enriched in hedgehog signaling (NES 2.05, adj p 0.005) and depleted in hematopoietic cell lineage (NES -2.24, adj p 0.001). The altered gene expression was also enriched in signatures with high-CpG density promoters bearing specific histone methylation marks (NES 2.05, adj p 0.002).

We identified potential pathologic mutations in signaling, epigenetic, and transcription factors including (Rock2, Ptprn, Ube4b, Kdm2, Aebp1, Ints1). ATAC-seq results identified differential chromatin states with binding motifs in the accessible chromatin regions suggesting hematopoietic transcription factors are altered at their binding sites (e.g. PU.1, IRF8). We demonstrate that Kdm2 loss in leukemia cell line K562 cells could blunt the expression of CD41 upon PMA induced megakaryocytic differentiation. This suggests genes regulating differentiation may mediate HMA resistance.

We screened a CRISPR epigenetic library of target genes that would inhibit growth in our Tet2 Flt3 leukemia model. We used an endothelial co-culture system to select target genes (guides that become depleted over a week in culture). We identified Brd2, Setdb1, Setd4, Kdm4a, Jmjd1c, Atad2b, and Chd6 as potential target genes. In vivo, we show that targeting of Kdm4 in Tet2 Flt3-mutant leukemia cells leads to reduced engraftment of leukemic cells compared to targeting a control locus or Ep300 (a positive selection gene) (mean engraftment, Ctrl:Ep300:Kdm4a 48%:54%:35%, p<.05). We tested pharmacologic inhibition of KDM4A and BRD2. We demonstrate that these agents could inhibit growth of leukemia cells including resistant clones and have combinatorial activity with venetoclax.

ConclusionTet2 Flt3 leukemia demonstrates a differentiation response to HMA therapy. Activation of this process may be important in the combination effect with venetoclax. When deriving resistant HMA clones, we identified genes in signaling, epigenetics, and transcriptional regulation that become mutated. These mutations affected the maturation of megakaryocyte-erythroid progenitors. Altered gene regulation at CpG islands may mediate these effects. CRISPR screening identified epigenetic targets (Brd2, Kdm4a)that have therapeutic potential in myeloidleukemia.