Genetic Interactions between Cohesin Mutations and Core-Binding Factor Driver Oncogenes

Introduction:

AML is a genetically heterogeneous disease, with an average 5-year survival of 50%. The core-binding factor complex is essential for normal hematopoiesis and is composed of two subunits, AML1 (aka RUNX1) and CBFB. Both AML1 and CBFB are involved in distinct chromosomal translocations in AML, t(8;21) and inv(16), which generate the fusion oncoproteins AML1-ETO or CBFB-MYH11 (Speck 2002). Heterozygous mutations in one of four members of the cohesin complex (RAD21, SMC3, STAG2, and SMC1A) are commonly found in patients with AML, and frequently (up to 25%) co-occur with AML1-ETO, but never with CBFB-MYH11 (Duployez 2016, Faber 2016). We hypothesize that cohesin mutations synergize with AML1-ETO during leukemic transformation, whereas CBFB-MYH11 and cohesin display a synthetic lethal genetic interaction.

Significance:

Patients with t(8;21) and inv(16) driven leukemia are treated identically in clinic, having the same prognosis and treatment strategy. Therefore, it is surprising that their co-mutational spectra are quite distinct. Identification of genetic cooperativity and/or synthetic lethality can yield valuable insights into the requisite steps required for AML development, thereby informing potential therapeutic options for these patients.

Aims:

We aim to determine the mechanism by which CBF driver-oncogenes form distinct genetic interactions with cohesin to promote AML.

Methods:

We have engineered murine-derived bone marrow cells that express AML1-ETO or CBFB-MYH11 and are either Smc3+/f or Smc3+/-. We have studied the phenotype of these cells in vitro and with next generation sequencing technologies.

Results:

Our in vitro studies indicate that the loss of cohesin augments the in vitro self-renewal of AML1-ETO, with increased self-renewal compared to AML1-ETO expression alone. By contrast, the introduction of Smc3+/- on the CBFB-MYH11 background reduced serial replating indicating a synthetic lethal interaction between the two.

Next, we performed molecular studies to identify the mechanisms underpinning the different phenotypes. First examining the AML1-ETO;Smc3+/- interaction, we performed ATAC-seq. In the AML1-ETO;Smc3+/- background, we have uncovered enrichment of several motifs implicated in myeloid development (RUNX1, GATA2, ERG, PU.1), nuclear architecture (CTCF, CTCFL), and cell proliferation (AP-1, FLI1, JUN). Additionally, RNA-seq reveals downregulation of genes involved in myeloid cell differentiation, changes corresponding to HoxA9 upregulation, and upregulation of the Rb and p53 oncogenic gene signatures in AML1-ETO;Smc3+/- compared to Smc3+/f. Results of our CBFB-MYH11 sequencing studies (RNAseq and ATACseq) are pending.

Conclusions:

We have identified that Smc3 haploinsufficiency and AML1-ETO cooperate to promote increased self-renewal in vitro while Smc3 haploinsufficiency and CBFB-MYH11 form a synthetic lethal interaction. Smc3 haploinsufficiency and AML1-ETO result in increased chromatin accessibility and transcriptional changes associated with a leukemic signature. Our findings lead us to propose that alteration of cohesin's function as a regulator of chromatin accessibility allows AML1-ETO to bind new sites leading to the transcriptional changes and our observed phenotype. Molecular mechanisms underlying the negative interaction between Smc3 haploinsufficiency and CBFB-MYH11 may be uncovered upon the completion of our NGS studies.

Future Directions:

We are currently performing animal studies, with both AML1-ETO and CBFB-MYH11 models, to identify an in vivo phenotype. To test our most recent AML1-ETO hypothesis, we plan to perform ChIP-seq to identify changes in genomic AML1-ETO binding sites that occur upon cohesin haploinsufficiency. Analysis of inv(16) NGS experiments expected to be completed within 3 months.