AstraZeneca’s WEE1 protein inhibitor AZD1775 Shows Success Against Tumors with a SETD2 mutation
Stephen J. Williams, Ph.D., Curator
There have been multiple trials investigating the utility of cyclin inhibitors as anti-tumoral agents (see post) with the idea of blocking mitotic entry however another potential antitumoral mechanism has been to drive the cell into mitosis in the presence of DNA damage or a defective DNA damage repair capacity. A recent trial investigating an inhibitor or the cell cycle checkpoint inhibitor Wee1 showed positive results in select cohorts of patients with mutations in DNA repair, indicating the therapeutic advantage of hijacking the cell’s own DNA damage response, much like how PARP inhibitor Olaparib works in BRCA1 mutation positive ovarian cancer patients.
John Carroll at FierceBiotech reported that an Oxford team spotlights promise of AstraZeneca drug in targeting cancers.
According to his report,
Investigators at Oxford University say that one of AstraZeneca’s ($AZN) pipeline drugs proved particularly effective in killing cancer cells with a particular genetic mutation.
The ex-Merck ($MRK) drug is AstraZeneca’s WEE1 protein inhibitor AZD1775, which proved particularly lethal to genes with a SETD2 mutation, which the researchers see as a potential ‘Achilles heel’ often found in kidney cancer and childhood brain tumors.
“When WEE1 was inhibited in cells with a SETD2 mutation, the levels of deoxynucleotides, the components that make DNA, dropped below the critical level needed for replication,” noted Oxford’s Andy Ryan. “Starved of these building blocks, the cells die. Importantly, normal cells in the body do not have SETD2 mutations, so these effects of WEE1 inhibition are potentially very selective to cancer cells.”
AstraZeneca landed rights to the drug back in 2013, when incoming Merck R&D chief Roger Perlmutter opted to spin it out while focusing an immense effort around the development of its PD-1 checkpoint inhibitor KEYTRUDA® (pembrolizumab). Since then, AstraZeneca has made it available to academic investigators through their open innovation program.
Since picking up the drug, AstraZeneca has posted positive mid-stage data for p53 mutated ovarian cancer at the last big ASCO meeting, (and see associated abstract on Multicenter randomized Phase II study of AZD1775 plus chemotherapy versus chemotherapy alone in patients with platinum-resistant TP53-mutated epithelial ovarian, fallopian tube, or primary peritoneal cancer) noting its qualification as a first-in-class player in their pipeline.
Wee1, DNA damage checkpoint and cell cycle regulation
In fission yeast, Wee1 delays entry into mitosis by inhibiting the activity of Cdk1, the cyclin-dependent kinase that promotes entry into mitosis (Cdk1 is encoded by the cdc2+ gene in fission yeast and the CDC28 gene in budding yeast) (Russell and Nurse, 1987a). Wee1 inhibits Cdk1 by phosphorylating a highly conserved tyrosine residue at the N-terminus (Featherstone and Russell, 1991; Gould and Nurse, 1989; Lundgren et al., 1991; Parker et al., 1992; Parker and Piwnica-Worms, 1992). The phosphatase Cdc25 promotes entry into mitosis by removing the inhibitory phosphorylation (Dunphy and Kumagai, 1991; Gautier et al., 1991; Kumagai and Dunphy, 1991; Millar et al., 1991; Russell and Nurse, 1986; Strausfeld et al., 1991). Loss of Wee1 activity causes cells to enter mitosis before sufficient growth has occurred and cytokinesis therefore produces two abnormally small daughter cells (Fig. 1A) (Nurse, 1975). Conversely, increasing the gene dosage of wee1 causes delayed entry into mitosis and an increase in cell size, indicating that the levels of Wee1 activity determine the timing of entry into mitosis and can have strong effects on cell size (Russell and Nurse, 1987a). Similarly, cdc25– mutants undergo delayed entry into mitosis, producing abnormally large cells, and an increase in the gene dosage of cdc25 causes premature entry into mitosis and decreased cell size (Russell and Nurse, 1986). Despite these difficulties, early work in fission yeast suggested that the Wee1 kinase plays an important role in a checkpoint that coordinates cell growth and cell division at the G2/M transition (Fantes and Nurse, 1978; Nurse, 1975; Thuriaux et al., 1978). WEE1 is an evolutionarily conserved nuclear tyrosine kinase (Table 2) that is markedly active during the S/G2 phase of the cell cycle [24, 25]. It was first discovered 25 years ago as a cell division cycle (cdc) mutant-wee1– in the fission yeast, Schizosaccharomyces pombe . Fission yeast lacking WEE1 are characterized by a smaller cell size, and this phenotype has been attributed to the ability of WEE1 to negatively regulate the activity of cyclin dependent kinase, Cdc2 (Cdc28 in budding yeast and CDK1 in human), in the Cdc2/CyclinB complex . Recently, WEE1 was shown to directly phosphorylate the mammalian core histone H2B at tyrosine 37 in a cell cycle dependent manner. Inhibition of WEE1 kinase activity either by a specific inhibitor (MK-1775) or suppression of its expression by RNA interference abrogated H2B Y37-phosphorylation with a concurrent increase in histone transcription .
As shown in the Below figure Wee1 is a CDK cyclin kinase which results in an inactivating phosphorylation event on CDK/Cyclin complexes
Figure 1. Schematic representation of the effects of Chk1 and Wee1 inhibition on CDK-CYCLIN complex regulation, that gets more activated being unphosphorylated from Cell cycle, checkpoints and cancer by Laura Carrassa.
Figure 2. Schematic representation of the role of Chk1 and Wee1 in regulation of the CDK-cyclin complexes involved in S phase and M phase entry from Cell cycle, checkpoints and cancer by Laura Carrassa.
The following articles discuss how Wee1 can be a target and synergize with current chemotherapy
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p53 mutation Frequency in Ovarian Cancer and contribution to chemo-resistance
The following is from the curated database TCGA and cBioPortal TCGA Data Viewer for mutations found in ovarian cancer sequencing studies in the literature
According to TCGA researchers have:
- Confirmed that mutations in gene TP53 are present in more than 96 percent of ovarian cases (>57% mutation frequency) while SETD2 mutations are present in only 1% of cases (1.1% mutation frequency).
In general, ovarian cancers with TP53 are considered to have increased resistance to commonly used cytotoxic agents used for this neoplasm, for example cisplatin and taxol, as TP53 is a major tumor suppressor/transcription factor involved in cell cycle, DNA damage response, and other chemosensitivity mechanisms. One subtype of TP53 mutations, widely termed gain-of-function (GOF) mutations, surprisingly converts this protein from a tumor suppressor to an oncogene. We term the resulting change an oncomorphism. In this review, we discuss particular TP53 mutations, including known oncomorphic properties of the resulting mutant p53 proteins. For example, several different oncomorphic mutations have been reported, but each mutation acts in a distinct manner and has a different effect on tumor progression and chemoresistance.
Figure 1. The spectrum of protection against cancer provided by WT p53. As copies of WT p53 (TP53+/+) are lost, cancer protection decreases. When oncomorphic mutations are acquired, cancer susceptibility is increased.
Oncomorphic p53 proteins were first identified over two decades ago, when different TP53 mutants were introduced into cells devoid of endogenous p53 [38,39]. Among all cancers, the most common oncomorphic mutations are at positions R248, R273, and R175, and in ovarian cancers the most common oncomorphic TP53 mutations are at positions R273, R248, R175, and Y220 at frequencies of 8.13%, 6.02%, 5.53%, and 3.74%, respectively [33,34]. In in vitro studies, cells with oncomorphic p53 demonstrate increased invasion, migration, angiogenesis, survival, and proliferation as well as resistance to chemotherapy [35,37,40,41].
Figure 2. Hotspots for TP53 mutations. Mutations that occur at a frequency greater than 3% are highlighted. Certain p53 mutants have oncomorphic activity (denoted by *), functioning through novel protein interactions as well as novel transcriptional targets to promote cell survival and potentially chemoresistance. Codons in the “other” category include those that produce non-functional p53 or have not been characterized to date.
Osman AA, Monroe MM, Ortega Alves MV, Patel AA, Katsonis P, Fitzgerald AL, Neskey DM, Frederick MJ, Woo SH, Caulin C, Hsu TK, McDonald TO, Kimmel M, Meyn RE, Lichtarge O, Myers JN.
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The protein phosphatase 2A inhibitor LB100 sensitizes ovarian carcinoma cells to cisplatin-mediated cytotoxicity.
- 1Laboratory of Cell Biology, Center for Cancer Research, National Cancer Institute, NIH, Bethesda, Maryland.
- 2Laboratory of Cancer Biology and Genetics, Center for Cancer Research, National Cancer Institute, NIH, Bethesda, Maryland.
- 3Surgical Neurology Branch, National Institute of Neurological Disorders and Stroke, NIH, Bethesda, Maryland.
- 4Laboratory of Cell Biology, Center for Cancer Research, National Cancer Institute, NIH, Bethesda, Maryland. email@example.com.
Despite early positive response to platinum-based chemotherapy, the majority of ovarian carcinomas develop resistance and progress to fatal disease. Protein phosphatase 2A (PP2A) is a ubiquitous phosphatase involved in the regulation of DNA-damage response (DDR) and cell-cycle checkpoint pathways. Recent studies have shown that LB100, a small-molecule inhibitor of PP2A, sensitizes cancer cells to radiation-mediated DNA damage. We hypothesized that LB100 could sensitize ovarian cancer cells to cisplatin treatment. We performed in vitro studies in SKOV-3, OVCAR-8, and PEO1, -4, and -6 ovarian cancer lines to assess cytotoxicity potentiation, cell-death mechanism(s), cell-cycle regulation, and DDR signaling. In vivo studies were conducted in an intraperitoneal metastatic mouse model using SKOV-3/f-Luc cells. LB100 sensitized ovarian carcinoma lines to cisplatin-mediated cell death. Sensitization via LB100 was mediated by abrogation of cell-cycle arrest induced by cisplatin. Loss of the cisplatin-induced checkpoint correlated with decreased Wee1 expression, increased cdc2 activation, and increased mitotic entry (p-histone H3). LB100 also induced constitutive hyperphosphorylation of DDR proteins (BRCA1, Chk2, and γH2AX), altered the chronology and persistence of JNK activation, and modulated the expression of 14-3-3 binding sites. In vivo, cisplatin sensitization via LB100 significantly enhanced tumor growth inhibition and prevented disease progression after treatment cessation. Our results suggest that LB100 sensitizes ovarian cancer cells to cisplatin in vitro and in vivo by modulation of the DDR pathway and cell-cycle checkpoint abrogation.
So Why SETD2 Mutations?
SETD2 is a histone methyltransferase that is specific for lysine-36 of histone H3, and methylation of this residue is associated with active chromatin and chromatin remodeling.
Evidences for mutations in the histone modifying gene SETD2 as critical drivers in leukemia development. Wang Q, et al. Sci China Life Sci, 2014 Sep. PMID 25077743
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Active NCI Clinical Trials of MK-1775 for Solid Tumors
NOTE Four Clinical Trials Investigating Mk-1775 and TP53 Status
|1||Recruiting||A Study of AZD1775 + Chemotherapy Versus Chemotherapy in Patients to Treat Ovarian, Fallopian Tube, Peritoneal Cancer.
|2||Recruiting||Gemcitabine Hydrochloride With or Without WEE1 Inhibitor MK-1775 in Treating Patients With Recurrent Ovarian, Primary Peritoneal, or Fallopian Tube Cancer
|3||Active, not recruiting||A Study of MK-1775 in Combination With Paclitaxel and Carboplatin Versus Paclitaxel and Carboplatin Alone for Participants With Platinum-Sensitive Ovarian Tumors With the P53 Gene Mutation (MK-1775-004)
|4||Not yet recruiting||Phase II, Single-arm Study of AZD1775 Monotherapy in Relapsed Small Cell Lung Cancer Patients
#2. Gemcitabine Hydrochloride With or Without WEE1 Inhibitor MK-1775 in Treating Patients With Recurrent Ovarian, Primary Peritoneal, or Fallopian Tube Cancer
This study is currently recruiting participants. (see Contacts and Locations)
ClinicalTrials.gov Identifier: NCT02101775
This randomized phase II clinical trial studies how well gemcitabine hydrochloride and WEE1 inhibitor MK-1775 work compared to gemcitabine hydrochloride alone in treating patients with ovarian, primary peritoneal, or fallopian tube cancer that has come back after a period of time. Gemcitabine hydrochloride may prevent tumor cells from multiplying by damaging their deoxyribonucleic acid (DNA, molecules that contain instructions for the proper development and functioning of cells), which in turn stops the tumor from growing. The protein WEE1 may help to repair the damaged tumor cells, so the tumor continues to grow. WEE1 inhibitor MK-1775 may block the WEE1 protein activity and may increase the effectiveness of gemcitabine hydrochloride by preventing the WEE1 protein from repairing damaged tumor cells without causing harm to normal cells. It is not yet known whether gemcitabine hydrochloride with or without WEE1 inhibitor MK-1775 may be an effective treatment for recurrent ovarian, primary peritoneal, or fallopian tube cancer.
Primary Outcome Measures:
- PFS evaluated using RECIST version 1.1 [ Time Frame: Time from start of treatment to time to progression or death, whichever occurs first, assessed up to 1 year ] [ Designated as safety issue: No ]
Secondary Outcome Measures:
- GCIG CA125 response rate [ Time Frame: Up to 1 year ] [ Designated as safety issue: No ]
- Incidence of grade 3 or 4 serious adverse events, graded according to the National Cancer Institute CTCAE version 4.0 [ Time Frame: Up to 1 year ] [ Designated as safety issue: Yes ]
- Objective response by RECIST version 1.1 [ Time Frame: Up to 1 year ] [ Designated as safety issue: No ]
- Overall survival [ Time Frame: Up to 1 year ] [ Designated as safety issue: No ]
Survival estimates will be computed using the Kaplan-Meier method.
- p53 protein expression in archival tumor tissue by immunohistochemistry (IHC) [ Time Frame: Baseline ] [ Designated as safety issue: No ]
- TP53 mutations (presence and type of mutation) by Sanger sequencing [ Time Frame: Baseline ] [ Designated as safety issue: No ]
These Trials Are Not Investigating TP53 Status of Patient Cohorts
A Phase I Study of Single-agent MK-1775, a Wee1 Inhibitor, in Patients With Advanced Refractory Solid Tumors
This study is currently recruiting participants. (see Contacts and Locations)
- To establish the safety and tolerability of single-agent MK-1775 in patients with refractory solid tumors
- To determine the pharmacokinetics of MK-1775 in patients with refractory solid tumors
- To determine the effect of MK-1775 on markers of DNA damage and apoptosis in tumor tissue and circulating tumor cells
- To evaluate the antitumor activity of MK-1775 in patients with refractory solid tumors
Note: A further expansion cohort of 6 additional patients with documented tumors harboring BRCA-1 or -2 mutations will lso be enrolled at the MTD to further explore the safety of the agent and obtain preliminary evidence of activity in this patient population
A Phase 1/2 Study of AZD1775 (MK-1775) in Combination With Oral Irinotecan in Children, Adolescents, and Young Adults With Relapsed or Refractory Solid Tumors
- To estimate the maximum tolerated dose (MTD) and/or recommended Phase 2 dose of MK-1775 (WEE1 inhibitor MK-1775) administered on days 1 through 5 every 21 days, in combination with oral irinotecan (irinotecan hydrochloride), to children with recurrent or refractory solid tumors.
- To define and describe the toxicities of MK-1775 in combination with oral irinotecan administered on this schedule.
III. To characterize the pharmacokinetics of MK-1775 in children with refractory cancer.
- To preliminarily define the antitumor activity of MK-1775 and irinotecan within the confines of a Phase 1 study.
- To obtain initial Phase 2 efficacy data on the anti-tumor activity of MK-1775 in combination with irinotecan administered to children with relapsed or refractory neuroblastoma and in children with relapsed or refractory medulloblastoma/CNS PNET (central nervous system primitive neuroectodermal tumor).
III. To investigate checkpoint over-ride by MK-1775 via the mechanism-based pharmacodynamic (PD) biomarker of decreased cyclin-dependent kinase 1 (CDK1) phosphorylation in correlative and exploratory studies.
- To evaluate potential predictive biomarkers of MK-1775 sensitivity, including v-myc avian myelocytomatosis viral oncogene homolog (MYC), v-myc avian myelocytomatosis viral oncogene neuroblastoma derived homolog (MYCN), phosphorylated-WEE1 G2 checkpoint kinase (p-Wee1), enhancer of zeste homolog 2 (Drosophila) (EZH2) and gamma-H2A histone family, member X (H2AX) in tumor tissues in correlative and exploratory studies.