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Mechanisms of Lineage Plasticity in Response to Therapy in AML
Sean Walulik1, Sarai Dean1, Mark Wunderlich1, Chih-Hsing Chou1, Kwangmin Choi, 1 Lee Grimes1, 2, Daniel Starczynowski1, 2, Linde Miles1, 2, and Andrew Volk1, 2.1 Cincinnati Children’s Hospital Medical Center, Cincinnati, USA
2 University of Cincinnati, Cincinnati, USA
Acute myeloid leukemia (AML) is an aggressive blood cancer driven predominantly by mutations in epigenetic regulators resulting in the rapid expansion of immature myeloid cells. AML arises and persists in a hostile environment where it needs to avoid both extrinsic (immune system, niche competition, chemotherapy) and intrinsic (replicative and oxidative stress) stressors to progress. It rapidly adapts to these factors via lineage plasticity using epigenetic reprogramming as its primary tool, making it exceedingly challenging to treat with chemotherapies. Venetoclax and Azacitidine (Ven/Aza) is increasingly the front-line therapy for AML, functioning through BH3 inhibition and DNA hypomethylation. This therapy (Ven/Aza hereafter) is generally well-tolerated and produces an improved response rate compared to other chemotherapies. However, all patients will eventually relapse with a highly aggressive refractory AML. Some recent studies suggest that monocytic character of the AML is responsible for resistance, but the latest evidence demonstrates that Ven/Aza can itself induce a lineage switch to monocytic character. In fact, these relapses tend to have monocytic character even if the original tumor was not monocytic, demonstrating the plastic potential of AML. This means a potent selection mechanism for plastic clones is likely driving resistance. The plasticity response is common to essentially all AMLs and is under-appreciated as a resistance mechanism. The epigenetic mechanisms of this process remain unclear and represent a significant barrier for therapy. Ven/Aza is widely used and is one of the most potent inducers of lineage plasticity in the AML therapy lineup, making it the ideal model to study therapy-driven plasticity in AML.
Here, we demonstrate that the driving force behind therapy resistance is the selection of highly lineage plastic immature populations that can undergo iterative epigenetic reprograming to escape therapy. Using a combination of single cell omics approaches in multiple human models, we uncovered that therapy induces hematopoietic stem cell-like character in these populations and unlocks them as potential novel differentiation nodes. In each case, lineage plasticity and epigenetic reprogramming in response to therapy was dependent on the repression of the Chromatin Assembly Factor 1 (CAF1) complex, a chromatin-bound master epigenetic regulator responsible for maintaining lineage fidelity through transcriptional regulation as a function of its role as a nucleosome assembly factor. When CAF1 is repressed, cells can change their fate because the chromatin becomes accessible to lineage fate-driving transcription factors. We found that in the case of AML, CAF1 repression and subsequent release from the chromatin by therapy allows for replacement with the pro-monocytic maturation factor PU.1 to access its target genes, leading to monocytic identity. This stepwise progression of the initial tumor through a lineage plastic stem-like state to a transcriptionally distinct relapse signature was mirrored in single cell data from bone marrow biopsies of AML patients undergoing Ven/Aza therapy.
We hypothesized that we could take advantage of this lineage plastic state induced by therapy-mediated CAF1 repression to drive maturation of the tumor. First, we needed to find a stimulable target that drives lineage maturation through activation of transcription factors. Based on our single cell data, we found that the plastic populations of cells expresses CSF3R (the G-CSF receptor) which when stimulated, can promote neutrophil maturation. While G-CSF stimulation alone was unable to drive maturation, when used in combination with Ven/Aza (or CAF1 depletion by shRNA against CHAF1B, the p60 subunit of CAF1) we were able to drive neutrophil differentiation in vitro and resolve tumors in vivo in each model tested. Together, these data suggest that Ven/Aza drives the formation of a targetable lineage plastic state in the leukemia, opening the door for new differentiation-based therapies.
This work is supported by R35GM142452, R00CA230314, and an ASH Junior Faculty Scholar Award.
Speakers
Andrew Volk