Inhibition of Wee1 is emerging as a novel therapeutic strategy for cancer, and some data suggest that cells with dysfunctional p53 are more sensitive to Wee1 inhibition combined with conventional chemotherapy than those with functional p53. We and others found that Wee1 inhibition sensitizes leukemia cells to cytarabine. Thus, we sought to determine whether chemosensitization by Wee1 inhibition is dependent on p53 dysfunction and whether combining Wee1 inhibition is tolerable and effective in vivo. Synergistic inhibition of proliferation with a Wee1 inhibitor in clinical development, MK1775, and cytarabine was observed in all acute myeloid leukemia (AML) cell lines tested, regardless of p53 functionality. Mechanistic studies indicate that inhibition of Wee1 abrogates the S-phase checkpoint and augments apoptosis induced by cytarabine. In AML and lung cancer cell lines, genetic disruption of p53 did not alter the cells' enhanced sensitivity to antimetabolites with Wee1 inhibition. Lastly, mice with AML were treated with cytarabine and/or MK1775. The combination of MK1775 and cytarabine was well-tolerated in mice and enhanced the anti-leukemia effects of cytarabine, including survival. Thus, inhibition of Wee1 sensitizes hematologic and solid tumor cell lines to antimetabolite chemotherapeutics, whether p53 is functional or not, suggesting that the use of p53 mutation as a predictive biomarker for response to Wee1 inhibition may be restricted to certain cancers and/or chemotherapeutics. These data provide preclinical justification for testing MK1775 and cytarabine in patients with leukemia.
The interaction of tumor necrosis factor-␣ with its receptor CD120a (p55) initiates downstream signaling cascades that include the activation of the mitogen-activated protein kinase (MAPK), p42 mapk/erk2 . The membrane proximal region of CD120a (p55) is Ser-, Thr-, and Pro-rich and contains four mitogen-activated protein kinase consensus phosphorylation sites. In recent work, we showed that CD120a (p55) itself is a target of phosphorylation by p42 mapk/erk2, and after phosphorylation, the receptor is redistributed from the cell surface and Golgi complex to intracellular tubular structures associated with elements of the endoplasmic reticulum. The goal of this study was to define the specific amino acid residues that are phosphorylated. Deletional mutagenesis of the cytoplasmic domain of CD120a (p55) indicated that two sites located between residues 207-254 and 250 -300 were phosphorylated predominantly on Thr and Ser residues, respectively. Site-directed mutagenesis of Ser and Thr residues contained within the extracellular signal-regulated kinase (ERK) consensus sequences indicated that the preferred residues were Thr-236 and Ser-270. Primary phosphorylation at these sites appeared to enable subsequent phosphorylation at Ser-240 and Ser-244, although the level of phosphorylation of these latter two sites was less than the preferred sites. Through the use of specific ligation of CD120a (p55) alone and mice deficient in CD120a (p55), CD120b (p75), or both receptors, CD120a (p55) was shown to be necessary and sufficient for the induction of kinase activity. These findings thus suggest that the phosphorylation of Thr-236 and Ser-270 within the membrane proximal region of CD120a (p55) are the preferred sites of phosphorylation by p42 mapk/erk2 and may set in motion phosphorylation at other sites.
While some children with acute lymphoblastic leukemia (ALL) have excellent prognoses, the prognosis for adults and children with T cell ALL is more guarded. Treatment for T-ALL is heavily dependent upon antimetabolite chemotherapeutics, including cytarabine. Targeted inhibition of WEE1 with AZD1775 has emerged as a strategy to sensitize cancer cells to cytarabine and other chemotherapeutics. We sought to determine if this strategy would be effective for T-ALL with clinically relevant anti-leukemia agents. We found that AZD1775 sensitizes T-ALL cells to several traditional anti-leukemia agents, acting synergistically with cytarabine by enhancing DNA damage and apoptosis. In addition to increased phosphorylation of H2AX at serine 139 (γH2AX), AZD1775 led to increased phosphorylation of H2AX at tyrosine 142, a signaling event associated with promotion of apoptosis over DNA repair. In a xenograft model of T-ALL, the addition of AZD1775 to cytarabine slowed leukemia progression and prolonged survival. Inhibition of WEE1 with AZD1775 sensitizes T-ALL to several anti-leukemia agents, particularly cytarabine. Mechanistically, AZD1775 promotes apoptosis over DNA repair in cells treated with cytarabine. These data support the development of clinical trials including AZD1775 in combination with conventional chemotherapy for acute leukemia.
Ligation of the tumor necrosis factor ␣ receptor CD120a initiates responses as diverse as apoptosis and the expression of NF-B-dependent pro-survival genes. How these opposing responses are controlled remains poorly understood. Here we demonstrate that phosphorylation by p42 mapk/erk2 inhibits the apoptotic activity of CD120a while preserving its ability to activate NF-B. Phosphorylated CD120a is re-localized from the Golgi complex to tubular structures of the endoplasmic reticulum wherein it recruits Bcl-2. Antisense-mediated down-regulation of Bcl-2 antagonized the localization of CD120a to tubular structures and reversed the protection from apoptosis conferred by receptor phosphorylation. We propose that phosphorylation of CD120a represents a novel, Bcl-2-dependent mechanism by which the apoptotic activity of the receptor may be regulated. Thus, oncogenic activation of p42 mapk/erk2 may serve to inhibit the apoptotic activity of this death receptor while preserving NF-B-dependent responses and may thus indirectly contribute to a failure to eliminate cells bearing oncogenes of the Ras-Raf-MEK-p42 mapk/erk2 pathway.
WEE1 is a cell cycle and DNA damage response kinase that is emerging as a therapeutic target for cancer. AZD1775 is a small molecule inhibitor of WEE1, currently in early phase clinical trials as a single agent and in combination with more conventional anti-neoplastic agents. As resistance to kinase inhibitors is frequent, we sought to identify mechanisms of resistance to WEE1 inhibition in acute leukemia. We found that AZD1775 resistant cell lines are dependent upon increased HDAC activity for their survival, in part due to increased KDM5A activity. In addition, gene expression analyses demonstrate HDAC dependent increase in MYC expression and c-MYC activity in AZD1775 treated resistant cells. Overexpression of c-MYC confers resistance to AZD1775 in cell lines with low baseline expression. Pharmacologic inhibition of BRD4, and thereby c-MYC, partially abrogated resistance to AZD1775. Thus, acquired resistance to WEE1 inhibition may be reversed by HDAC or BRD4 inhibition in leukemia cells.
Phosphorylation of the TNF-alpha receptor TNF-R1 has been shown to differentially regulate receptor signaling and function and promote changes in its subcellular localization. Previous studies have shown that p42(mapk/erk2) phosphorylates Ser and Thr residues (T236, S240, S244, and S270) in the membrane proximal region of TNF-R1 and that mutation of these residues to Glu and Asp residues (TNF-R1.4D/E) mimics the effect of phosphorylation on receptor signaling and localization. In the present study, we investigated whether the initial phosphorylation of these residues by p42(mapk/erk2) promotes hierarchical phosphorylation of additional sites within the cytoplasmic domain of TNF-R1. This question was addressed by investigating the ability of the TNF-R1.4D/E mutant receptor to be phosphorylated in in vitro kinase assays using GST-mutant cytoplasmic domain fusion proteins as substrates and in intact cells following mutant receptor expression. In addition, we determined the location of the additional phosphorylation sites. Incubation of Sepharose bead-bound GST-TNF-R1(207)(-)(425).4D/E fusion protein with lysates containing activated p42(mapk/erk2) led to the phosphorylation of Ser and Thr residues in addition to the previously defined sites at T236, S240, S244, and S270. Deletional mutagenesis localized these residues to a stretch of 14 amino acids that encompasses three basic Pro-directed ([S/T]P) kinase consensus sequences located between residues S256 and T267. Point mutagenesis of T257, S262, and T267 to Ala residues indicated that these sites are targets of phosphorylation by p42(mapk/)(erk2). These findings support the conclusion that p42(mapk/erk2) promotes extensive phosphorylation of the membrane proximal region in a hierarchical fashion at both consensus and nonconsensus ERK-phosphorylation sites.
<p>PDF - 1164KB, Supplemental Table 1: Inhibition of Wee1 inhibits proliferation of AML cells independent of known molecular abnormalities. Supplemental Figure 1: AML cells are sensitive to MK1775 independent of p53 functionality. Supplemental Figure 2: MK1775 sensitizes AML cells to cytarabine independent of p53 function. Supplemental Figure 3: MK1775 sensitizes AML cells to cytarabine independent of p53 function. Supplemental Figure 4: MK1775 inhibits phosphorylation of CDK1, but does not cause widespread unscheduled mitosis. Supplemental Figure 5: Treatment with MK1775 does not sensitize AML cells to doxorubicin. Supplemental Figure 6: Treatment of AML cells with cytarabine and MK1775 is synergistic independent of p53 function in an isogenic model. Supplemental Figure 7: Treatment of lung cancer cells with antimetabolites and MK1775 is synergistic. Supplemental Figure 8: MK1775 and cytarabine is more effective than cytarabine alone in vivo.</p>
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