An unintended late-effect of the use of chemotherapy and/or irradiation in cancer treatment is the development of therapy-related myeloid neoplasms (t-MN). T-MN are associated with high-risk cytogenetic abnormalities and a poor prognosis of less than one year survival. The most common cytogenetic change in t-MN is the loss of chromosome 7 (-7) or the long arm, del(7q), occurring in almost half of patients and associated with alkylating agent exposure. Del(7q), or inactivating mutations of the CUX1 transcription factor encoded on 7q, have been reported to exist prior to exposure to genotoxic therapy in the form of clonal hematopoiesis of indeterminate potential (CHIP). How -7/del(7q) contributes to the pathogenesis of t-MN remains unclear.
We previously reported two inducible, shRNA-transgenic, CUX1-knockdown mouse lines: Cux1-mid (~50% residual CUX1) and Cux1-low (~12% residual CUX1). Both models develop myelodysplastic syndrome (MDS), however only Cux1-low mice have increased mortality (median survival = 275 days). To model t-MN, we treated these mice with an alkylating agent, N-ethyl-N-nitrosourea (ENU). While no ENU-treated control mice developed myeloid malignancies (n=16, median survival = 320 days), 43% of Cux1-mid (n=7, median survival = 160 days) and 91% of Cux1-low mice (n=11, median survival = 124 days) developed myeloid malignancies. Disease onset in ENU-treated CUX1-knockdown mice was significantly faster than in non-ENU-treated counterparts. Myeloid malignancies in ENU-treated CUX1-knockdown mice included acute myeloid leukemia and MDS. To our knowledge, this is the most rapid and penetrant murine model of t-MN reported.
CUX1-knockdown hematopoietic stem and progenitor cells (HSPCs) have increased proliferation and outcompete wild-type competitors in the setting of regenerative hematopoiesis. After ENU, CUX1-deficientcells have an additional, and significant, fitness advantage, rapidly outcompeting wild-type counterparts in the peripheral blood in competitive bone marrow transplants. Post-ENU, CUX1-knockdown HSPCs have higher levels of BrdU incorporation, indicating that CUX1-deficiency promotes continued HSPC proliferation after genotoxic stress. RNA-seq post-ENU exposure revealed that, compared to control LSKs (lin-/sca1+/ckit+), CUX1-knockdown LSKs have decreased expression of DNA repair and G2/M checkpoint pathways. To determine the role for CUX1 in the DNA damage response, we assessed γH2AX by flow cytometry after irradiation of HSPCs in vitro. CUX1 deficiency significantly blunted H2AX phosphorylation. Comet tail assays after in vivo irradiation demonstrated increased DNA damage at 6 and 24 hours post-treatment in CUX1-deficient HSPCs. These findings are consistent with failure to properly recognize DNA damage and progression through the G2/M checkpoint with unrepaired DNA damage in CUX1-deficient HSPCs. Overall, our studies indicate that CUX1 deficiency promotes clonal hematopoiesis post-alkylator treatment, impairs the HSPC DNA damage response, and predisposes to myeloid transformation in t-MN. In addition, loss of a single 7q gene, CUX1, is sufficient to predispose mice to t-MN development.
Disclosures
No relevant conflicts of interest to declare.
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