DNA Damage Repair Interference By WEE1 Inhibition with AZD1775 Overcomes Combined Azacitidine and Venetoclax Resistance in Acute Myeloid Leukmeia (AML)

Acute myeloid leukemia (AML) has remained one of the most treatment resistant and deadliest cancers. The survival of AML blast cells is controlled by the balance of anti- and pro-apoptotic proteins. Recently approved Bcl-2 targeted therapy of AML with the Bcl-2 specific inhibitor Venetoclax in combinations has improved patients outcomes. However, a priori and developing resistance to venetoclax combinations with hypomethylating agents (HMA) azacitidine and decitabine challenge this treatment. As such, novel therapies to overcome venetoclax-HMA resistance are urgently needed. We have identified a combination of DNA damage repair interference by WEE1 inhibition with AZD1775, combined with low dose cytarabine (AraC) as an effective strategy to overcome combined venetoclax-azacitidine resistance (VAR). AZD1775 with low dose AraC induced massive apoptosis (by Annexin V and cleaved caspase-3) and almost completely reduced viability and clonogenic growth of primary AML cells. To delineate the molecular mechanism of the synergistic effect of AZD1775/AraC we performed RNAseq analysis of single agent or the combination of AZD1775+AraC in AML cell lines and primary CD34+ selected AML patient cells with the goal to identify deferentially regulated genes indicating a mechanistic underpinning of the potent activity. Only 2 genes were deferentially regulated across cell lines and CD34+ selected cells under AZD1775+AraC treatment: one of these is NR4A1, an orphan nuclear receptor, which we went on to validate as a potential downstream target of Wee1 inhibition. The inactivation of NR4A1 in mice was previously shown to induce AML and to maintain leukemia stem cells. Using qPCR we confirmed that the expression of NR4A1 is upregulated after AZD1775/AraC combo treatment in human leukemic cells. We then demonstrated that activators of NR4A1 (cytosporone B and pPhOCH3) reduce viability of leukemic cells, while NR4A1 inhibitor pPhOH was able to abolish the effect of AZD1775/AraC combo treatment increasing leukemic cell viability]. To investigate the involvement of mitochondria in the effect of AZD1775/AraC treatment we performed the expression of mitochondrial genes and pathway analyses in RNAseq data and found that mitochondrial gene expression, including many genes involved in apoptosis, has most dramatic changes in the combo treatment if compared to the single agents. Subsequently, we have examined the expression of the main BCL-2 family apoptotic genes by qPCR and western bl