Oncogenic RAS promotes leukemic transformation of CUX1-deficient cells.

-7/del(7q) is prevalent across subtypes of myeloid neoplasms. CUX1, located on 7q22, encodes a homeodomain-containing transcription factor, and, like -7/del(7q), CUX1 inactivating mutations independently carry a poor prognosis. As with loss of 7q, CUX1 mutations often occur early in disease pathogenesis. We reported that CUX1 deficiency causes myelodysplastic syndrome in mice but was insufficient to drive acute myeloid leukemia (AML). Given the known association between -7/del(7q) and RAS pathway mutations, we mined cancer genome databases and explicitly linked CUX1 mutations with oncogenic RAS mutations. To determine if activated RAS and CUX1 deficiency promote leukemogenesis, we generated mice bearing NrasG12D and CUX1-knockdown which developed AML, not seen in mice with either mutation alone. Oncogenic RAS imparts increased self-renewal on CUX1-deficient hematopoietic stem/progenitor cells (HSPCs). Reciprocally, CUX1 knockdown amplifies RAS signaling through reduction of negative regulators of RAS/PI3K signaling. Double mutant HSPCs were responsive to PIK3 or MEK inhibition. Similarly, low expression of CUX1 in primary AML samples correlates with sensitivity to the same inhibitors, suggesting a potential therapy for malignancies with CUX1 inactivation. This work demonstrates an unexpected convergence of an oncogene and tumor suppressor gene on the same pathway.

Loss of a 7q gene, CUX1, disrupts epigenetically driven DNA repair and drives therapy-related myeloid neoplasms.

Therapy-related myeloid neoplasms (t-MNs) are high-risk late effects with poorly understood pathogenesis in cancer survivors. It has been postulated that, in some cases, hematopoietic stem and progenitor cells (HSPCs) harboring mutations are selected for by cytotoxic exposures and transform. Here, we evaluate this model in the context of deficiency of CUX1, a transcription factor encoded on chromosome 7q and deleted in half of t-MN cases. We report that CUX1 has a critical early role in the DNA repair process in HSPCs. Mechanistically, CUX1 recruits the histone methyltransferase EHMT2 to DNA breaks to promote downstream H3K9 and H3K27 methylation, phosphorylated ATM retention, subsequent γH2AX focus formation and propagation, and, ultimately, 53BP1 recruitment. Despite significant unrepaired DNA damage sustained in CUX1-deficient murine HSPCs after cytotoxic exposures, they continue to proliferate and expand, mimicking clonal hematopoiesis in patients postchemotherapy. As a consequence, preexisting CUX1 deficiency predisposes mice to highly penetrant and rapidly fatal therapy-related erythroleukemias. These findings establish the importance of epigenetic regulation of HSPC DNA repair and position CUX1 as a gatekeeper in myeloid transformation.

Gene dosage effect of CUX1 in a murine model disrupts HSC homeostasis and controls the severity and mortality of MDS.

Monosomy 7 (-7) and del(7q) are high-risk cytogenetic abnormalities common in myeloid malignancies. We previously reported that CUX1, a homeodomain-containing transcription factor encoded on 7q22, is frequently inactivated in myeloid neoplasms, and CUX1 myeloid tumor suppressor activity is conserved from humans to Drosophila. CUX1-inactivating mutations are recurrent in clonal hematopoiesis of indeterminate potential as well as myeloid malignancies, in which they independently carry a poor prognosis. To determine the role for CUX1 in hematopoiesis, we generated 2 short hairpin RNA-based mouse models with ∼54% (Cux1mid) or ∼12% (Cux1low) residual CUX1 protein. Cux1mid mice develop myelodysplastic syndrome (MDS) with anemia and trilineage dysplasia, whereas CUX1low mice developed MDS/myeloproliferative neoplasms and anemia. In diseased mice, restoration of CUX1 expression was sufficient to reverse the disease. CUX1 knockdown bone marrow transplant recipients exhibited a transient hematopoietic expansion, followed by a reduction of hematopoietic stem cells (HSCs), and fatal bone marrow failure, in a dose-dependent manner. RNA-sequencing after CUX1 knockdown in human CD34+ cells identified a -7/del(7q) MDS gene signature and altered differentiation, proliferative, and phosphatidylinositol 3-kinase (PI3K) signaling pathways. In functional assays, CUX1 maintained HSC quiescence and repressed proliferation. These homeostatic changes occurred in parallel with decreased expression of the PI3K inhibitor, Pik3ip1, and elevated PI3K/AKT signaling upon CUX1 knockdown. Our data support a model wherein CUX1 knockdown promotes PI3K signaling, drives HSC exit from quiescence and proliferation, and results in HSC exhaustion. Our results also demonstrate that reduction of a single 7q gene, Cux1, is sufficient to cause MDS in mice.