Differential Response to ATR/Chk1 Inhibition in Preleukemia and Transformed Leukemia in an MLL-ENL Model of Leukemogenesis Reflects Enrichment for Myc-Transcriptional Program

We study a dual role of ATR/Chk1 signaling in preleukemia and fully transformed AML and their vulnerability to specific inhibitors (i). We previously reported oncogene-inducible Mll-ENL mouse model, with AML evolving from preleukemia after a long latency period (Takacova et al, PMID: 22516260), where the activation of ATR/Chk1- and ATM/Chk2-mediated checkpoints contributed to an intrinsic DDR barrier preventing full leukemia development. Experimental suppression of this barrier by ATR/ATMi caffeine promoted the progression of preleukemia to leukemia. We hypothesize that in preleukemia, ATR/Chk1 DNA damage response (DDR) checkpoint activation serves as part of intrinsic anti-cancer barrier, while in leukemia, ATR-dependent signaling is essential for leukemia cell proliferation and survival.

We tested the consequences of ATR/Chk1 inhibition during Mll-ENL leukemogenesis, using ceralasertib (ATRi1, p.o. 25 mg/kg), elimusertib (ATRi2, p.o. 50 mg/kg) and AZD-7762 (Chk1i, i.v. 25 mg/kg), 3-5 times weekly, 1-6 months, using the preleukemic mice. We also created a cell culture model (MEER) by obtaining c-kit⁺ cells from bone marrow (BM) of Mll-ENL mice and adapting these cells to FLT3 ligand for growth.

Transcriptome of cells isolated from spleens of Mll-ENL mice during early preleukemia, late preleukemia (progression) and transformed leukemia revealed an enrichment for E2F target genes in early preleukemia. The DNA repair program was significantly activated in the late preleukemia stage, with the beginning of switch from E2F to Myc signature. Ambra1, a regulator of G1/S phase transition and genomic integrity in tumorigenesis (Maiani et al, PMID: 33854232), was downregulated compared to early preleukemia. In transformed leukemia, the most significantly enriched gene set was the Myc transcriptome; Ambra1 levels remained low compared to early preleukemia, implicating therapeutic ATR/Chk1 targeting at this stage (Murga et al, PMID: 22120667; Maiani et al).

Provision of the milder ATRi1 to preleukemic mice led to an increase of immature c-kit⁺/Mac-1⁺ cells in BM and spleen, increased rate of leukemic transformation and shorter survival. Administration of Chk1i also resulted in an increase of the immature myeloid cell subset. Consistently, our data revealed the weakening of antiproliferative barrier as a result of DDR signaling attenuation by ATR/Chk1 inhibition. More potent ATRi2 revealed in vivo toxicity to hematopoiesis, but did not specifically eliminate preleukemia Mll-ENL cells.

We then tested whether targeting of the DDR pathway in the late preleukemia, promoting proliferation of immature Mll-ENL cells, has the potential to induce specific synthetic lethality with oncogenic kinase inhibition. In vitro, MEER cells showed high sensitivity to JAK2i ruxolitinib (RX, IC50 24 nM), higher than to the tested FLT3i. Addition of RX (p.o. 90 mg/kg, 5 times weekly, 3 months) to caffeine administration in the late preleukemia did not change the rate of transformation into AML. Exposing transformed Mll-ENL myeloid progenitors to RX ex-vivo did not affect their survival or plating efficiency, only partially decreased cellularity of colonies in high RX concentrations.

Finally, an in-vivo model of transformed AML was created by serial transplantation of MEER cells into SCID mice. Tertiary recipients were treated with the above-mentioned DDR inhibitors/doses. Only ATRi2 significantly extended overall survival compared to ATRi1, Chk1i or vehicle treatment in transformed leukemic mice. We observed the induction of robust DNA damage, resulting in leukemia cells failing to proceed through the cell cycle and arresting in the S phase or dying.

In summary, we show differential response toATRi/Chk1i in preleukemia vs. fully transformed AML, reflecting threshold of DDR signaling (Bartek et al, PMID: 22218289) and extent of enrichment for Myc-driven transcriptional program. The suprathreshold levels of DNA damage and cell death was achieved only in Myc-driven transformed cells. In preleukemia, attenuation of ATR/Chk1 checkpoint promotes the development of leukemia from preleukemia. Combinatory targeting of DDR components and activated oncogenic signaling to induce synthetic lethality in preleukemia stage of MLL remains to be fully elucidated.

Grant support: Czech grant agencies projects NU21-03-00338, GACR-24-11730S, LX22NPO5102, JG_2023_016.

P. C. and L. J. share co-first authorship.

No relevant conflicts of interest to declare.