ETS1 Recruits the Canonical Baf Complex to Reactivate the Hematopoietic Stem Cell MYB Enhancer in the T-Cell Leukemia Context

In T-cell acute lymphoblastic leukemia (T-ALL), Notch variants are the most common oncogenic mutations, but clinical trials showed excessive toxicity of pan-Notch inhibitors. In response, we refocused to ETS1, which we and others previously showed is the top transcription factor that most frequently co-binds Notch-occupied elements in T-ALL. Here, we performed an unbiased genome-wide CRISPR-interference screen of the strongest ETS1-dependent enhancers. The #2 ranked element was the Notch-MYC enhancer (N-Me). The #1 ranked element was a +140kb Notch-bound enhancer interacting with the MYB promoter that we named the “ETS-MYB enhancer” or E-Me. MYB is an oncogene across all T-ALL subgroups. Chromatin profiling showed that the E-Me is highly active and accessible in hematopoietic stem cells (HSCs) and then silenced during differentiation. In contrast, the E-Me is highly active in Notch-type T-ALL. Therefore, we hypothesized that the E-Me is a HSC enhancer that is reactivated in T-ALL cells to drive MYB expression and promote population cell growth.

To test this, we first determined the physiological role of the E-Me in hematopoietic cells using a novel E-Me conditional knockout mouse. E-Me deletion reduced Myb expression in HSCs 2-fold and increased absolute numbers 2.1-fold, whereas other hematopoietic populations were minimally affected. E-Me-deficient HSCs were defective as they reconstituted poorly in serial competitive bone marrow transplants and were depleted 6.2-fold in aged mice. Lastly, germline E-Me inactivation had no long-term effects on mouse weight or survival. These data suggest that the E-Me has limited function in committed T-cell progenitors and normal physiology but is important for HSC long-term self-renewal.

We next wanted to examine E-Me function in murine and human T-ALL leukemogenesis. To do this, we first generated Notch-induced and Lmo2-induced T-ALL mouse models. E-Me deletion during initiation or maintenance reduced blast counts 38-to-105 fold and significantly prolonged survival. Next, we transduced E-Me sgRNAs into human T-ALL cell lines, which suppressed MYB expression 3.3-to-5.3-fold and inhibited cell proliferation 17-to-113-fold. These results indicate that the E-Me has major importance in murine and human leukemic cell growth and MYB induction.

Following this, we wanted to understand how ETS1 induces E-Me activity. We first performed HOMER analysis of the E-Me and identified a single conserved ETS1-binding motif. Mutating this site decreased pulldown of ETS1 but no other transcription factor in reverse ChIP mass spectrometry and abrogated E-Me activity in reporter assays. In contrast, Notch inhibitors had no effect. Next, we generated a mouse model with mutation of the ETS1 site. Sequencing of ETS1 ChIP pulldowns confirmed the inability of ETS1 to bind the mutated motif. These mutant mice showed no significant alterations during steady state thymopoiesis but had impaired T-cell regeneration after sublethal irradiation. Dual reverse ChIP and co-IP mass spectrometry screens in a T-ALL cell line identified cBAF, a chromatin remodeling complex, as a top ranked ETS1 cofactor that is recruited to the E-Me. To confirm this, we genetically degraded ETS1 and found 2.5-fold reduced cBAF occupancy, 3.2-fold reduced H3K27ac signals, and 4.9-fold reduced ATAC-seq signals at the E-Me. ETS1 deprivation also reduced occupancy of Notch-associated transcriptional regulators. Lastly, AU-15330, a PROTAC degrader of cBAF, impaired E-Me H3K27ac signals 2.1-11.1-fold and reduced MYB protein levels 2.3–9.7-fold. These data suggest that ETS1 recruits cBAF after transformation to promote chromatin accessibility at the E-Me.

There is an unmet need to identify the most important oncogenic enhancers and find ways to safely eject transcription factors bound to these elements as potential therapies. In addressing this, our unbiased screen revealed the top importance of a stem cell enhancer, the E-Me, which was ranked higher than the N-Me. We also suggest ways to inactivate the E-Me with cBAF and ETS1 degraders, which mouse studies predict would be safer than pan-Notch or pan-MYB inhibition. Finally, the literature provides ample examples of Notch having a central role in T-ALL. In contrast, we identify a top-ranked oncogenic enhancer that is independent of Notch but requires ETS1 to remodel chromatin to enable transcription factor complex assembly and function.