The principal aim of this study was to investigate the efficacy of MK-1775 as a single agent and in combination with GEM in PDA xenografts and to assess whether the status of the p53 gene had any role in dictating the efficacy of the treatment. We used a xenograft model which is freshly generated from the tumors taken from pancreatic cancer patients and selected nine xenografts (six xenografts with p53-deficient and three xenografts with p53-wild type status) for this study. As shown in , tumors in vehicle-treated animals grew rapidly. Single agent MK-1775 treatment produced greater than 50% inhibition of tumor growth in two xenografts (PANC286 and PANC198). However, five of nine xenografts treated with GEM and six of nine xenografts treated with GEM plus MK-1775 produced complete tumor growth inhibition and in fact resulted tumor shrinkage compared to control and MK-1775 treated animals (). These data suggest that single agent MK-1775 is unlikely to be effective in patients with PDA but that the combination of this agent with GEM has a substantial level of activity, and should be prioritized for clinical development.
Combination of MK-1775 and gemcitabine potentiates the efficacy of gemcitabine in established human pancreatic cancer xenografts
Overall, none of the xenografts with p53-deficient status in the GEM treatment group produced 50% regression of initial tumor volume (). However, combination of GEM and MK-1775 resulted in greater than 50% regression of initial tumor volume in four of six xenografts (66.66%) with p53-deficient status (). The number of tumors that regressed more than 50% of its initial tumor size in each xenograft upon completion of treatment is provided in . Tumors with wild type-p53 status did not regress with treatments (). Among the xenografts with p53-deficient status, GEM alone treatment induced regression in 7 of 55 tumors (12.72%), while MK-1775 in combination with GEM induced regressions in 25 of 49 tumors (51.10%) in the six xenografts. Tumor growth regressions in GEM plus MK-1775 treated mice were found to be significant in PANC198 (P
< 0.0001), PANC215 (P
< 0.005) and PANC185 (P
< 0.005) as compared to GEM treated mice. There was an overall 4.01 fold increase in total number of tumors regressed in the combination treatment group compared to GEM alone treatment (). K-ras
status do not influence the tumor regression pattern in the xenografts (). One limitation of preclinical studies is that the threshold of activity that translates into positive clinical outcome is not known. Often, drugs are selected for clinical development base on tumor growth inhibition in preclinical models. As our experience with freshly generated PDA models increases and more comparison data is available, we are observing that indeed only agents that result in marked tumor regressions in this model have the potential to impact patient outcome. This is illustrated by our recent work on AZD0530 and nab-paclitaxel. AZD0530, a Src kinase inhibitor, induced only modest inhibition of tumor growth in PDA xenografts and, as expected, failed in a phase II clinical trial (27
). In contrast, nab-paclitaxel, in combination with GEM, resulted in marked tumor regression in this model, which successfully predicted a positive phase II study (29
Mutational status and number of tumors regressed morethan 50% of initial size as on day 28
The selective augmentation of antitumor effects in tumors with deficient-p53 was anticipated based on the mechanism of action of the agent. Mammalian cells undergo cell cycle arrest in response to DNA damage due to the existence of multiple checkpoint response mechanisms. In response to DNA damage, the cell cycle halts, preventing the propagation of cells with damaged DNA. DNA damage culminates in the enforcement of cell cycle arrest, mainly at G1 and G2 phases. Checkpoint pathways operating at the G1 phase are frequently lost in cancer cells due to mutation of the p53 tumor suppressor gene. Cells lacking functional p53 would not be anticipated to arrest at the G1 checkpoint and would depend on the G2 checkpoint to permit DNA repair prior to undergoing mitosis. Thus, G2 checkpoint abrogation should preferentially kill p53-deficient cancer cells by removing the only checkpoint that protects these cells from premature entry into mitosis in response to DNA damage. Our data strongly suggest that the clinical development of MK-1775 with GEM should be restricted to patients with p53-deficient PDA.
Cdc2 initiates mitosis, which is the ultimate target of DNA replication and repair checkpoints. Chk1, Chk2, Wee1, and Myt1 are key regulators of G2
checkpoint, which act directly or indirectly to inhibit Cdc2 activity (17
). Chk1 and Chk2 are downstream effectors of ataxia telangiectasia-mutated kinase (ATM) and ataxia telangiectasia and Rad3-related kinase (ATR), which induce G2
/M cell cycle arrest by inactivating Cdc25 tyrosine phosphatases through phosphorylation (32
). Both Chk1 and Chk2 are known to phosphorylate Cdc25 on Ser216 and this phosphorylation makes Cdc25 functionally inactive (33
). Cdc25 is required for removal of inhibitory phosphotyrosines on Cdc2/cyclin B1 kinase complexes that mediate entry into mitosis. On the other hand, the inhibitory phosphorylations at Thr-14 and Tyr-15 sites of Cdc2 are mediated by Myt1 and Wee1 kinases (34
). Wee1 is the major kinase phosphorylating the Tyr-15 site and Wee1 dependent phosphorylation of Cdc2 maintains the Cdc2/cyclin B1 complex in an inert form. While Myt1 preferentially phosphorylates the Thr-14 site, it can also phosphorylate the Tyr-15 site. Thus either Cdc25 inactivation and/or Wee1/Myt1 activation could contribute to G2
cell cycle arrest in response to DNA damage.
Chk1/2 inhibitors are in clinical development (36
). A recent report indicated that Chk1 is required to maintain genome integrity and cell viability, and that p53-wild type cells are no less sensitive than p53-deficient cells to Chk1 inhibition in the presence of DNA damage. Thus, combining Chk1 inhibition with DNA damaging agents does not lead to preferential killing of p53-deficient over p53-wild type cells, and inhibiting Chk1 does not appear to be a promising approach for potentiation of cancer chemotherapy (39
). Here we showed that Wee1 inhibition by MK-1775 could potentiate GEM sensitivity and tumor regressions, selectively in p53-deficient pancreatic cancer xenografts.
We were also interested in long-term tumor growth control and followed three xenografts after treatment for an extended period of time. Tumor re-growth data, as shown in suggest that not only does the combination of GEM with MK-1775 lead to synergistic tumor growth inhibition, but the effect of the combination therapy is also longer-lasting than that seen with GEM alone (). It was noteworthy, however, that tumors eventually recur, albeit at a slower pace.
MK-1775 synergize with gemcitabine to inhibit tumor growth of human pancreatic cancer xenografts
In order to determine the target modulation by MK-1775, we examined Wee1, Cdc2 and their phosphorylated forms in post treatment tumor specimens. MK-1775 treatment strongly inhibited phosphorylation of Tyr-15 of Cdc2, the primary substrate of Wee1 (, ), suggesting increased Cdc2 kinase activity. In addition, the Wee1 protein was consistently reduced by MK-1775 treatment as shown by western blotting (), likely due to degradation of Wee1 as MK-1775 treatment activates Cdc2 which in turn phosphorylates Wee1, ultimately leading to its ubiquitin-proteasome dependent destruction (40
Combination of MK-1775 and gemcitabine inhibits Wee1 and attenuates Cdc2 phosphorylation to promote mitotic entry and apoptosis
Immunohistochemical staining of phospho-Cdc2 and phospho-histone H3
To determine whether combination therapy promotes mitotic entry, we measured the expression of phospho histone H3 by western blot as well as by immunohistochemistry. When administered in combination with GEM, MK-1775 promoted mitotic entry as measured by enhanced phospho histone H3 expression (, ). In addition, the combined treatment resulted in the up-regulation of C-PARP as well as down regulation of cIAP2, suggesting that combination therapy facilitates apoptotic death of tumor cells (). GEM, as a chain terminator, requires an active cell cycle to be effective for inhibiting tumor growth, and might induce cell cycle halt and enforce cell cycle checkpoints, which may play an important role in escalating the resistance to therapy. Thus, there is a strong rationale in combining checkpoint inhibitors with GEM as a means to enhance tumor response (41
). Here we showed that GEM induces G2
arrest, which correlates with an increased Cdc2 inhibitory phosphorylation at Tyr-15 and prevents mitotic entry as evidenced by decreased p-HH3Ser10
(). However, the decreased Cdc2 inhibitory phosphorylation at Tyr-15 caused by MK-1775 treatment indicates that MK-1775 has the ability to abrogate the G2
arrest induced by GEM and promote mitotic entry as demonstrated by enhanced phospho histone H3 at Ser-10 (). Cyclin B1 was examined as a marker of G2 phase (43
). Expression of Cdc2 was not altered by treatments, while the expression of Cyclin B1 was strongly inhibited by MK-1775 as well as combination of MK-1775 and GEM treatment compared to control and GEM treated tumors of PANC198 (). Loss of Cyclin B1 accumulation in the MK-1775 as well as combination of MK-1775 and GEM treated tumors indicate the exit from G2 phase arrest (). The levels of γ-H2AX were used as a surrogate for unrepaired DNA damage (44
). γ-H2AX expression was clearly elevated in the combination of MK-1775 and GEM treatment group compared to GEM treated tumors of PANC198, indicating the persistence of unrepaired DNA damage in the tumors (). Overall, in addition to providing mechanistic support to the observations made above, the data provides important clues for potential biomarkers for clinical development of this drug combination.
In conclusion, our results provide compelling evidence that MK-1775 treatment leads to the inhibition and subsequent loss of Wee1 and activation of its substrate, Cdc2. The MK-1775 and GEM combination promoted the mitotic entry of tumor cells and eventually led to apoptotic death, and delayed the tumor progression compared to the GEM treatment. These findings have important clinical implications and raise the hope for potential therapeutic benefit to many PDA patients whose cancer cells are deficient for p53 function.