Abstract A33: Defect in S phase cell cycle checkpoint renders tumours vulnerable to CHK1 inhibitor single-agent treatment in vitro and in vivo

Mutations in TP53 occur commonly in the majority of human tumors and confer aggressive tumor phenotypes including metastasis and therapy resistance. CB002 and structural-analogues restore p53 signaling in tumors with mutant-p53 but we find that unlike other xanthines such as caffeine, pentoxifylline, and theophylline, they do not deregulate the G2-checkpoint. Novel CB002-analogues induce pro-apoptotic Noxa protein in an ATF3/4-dependent manner, whereas caffeine, pentoxifylline, and theophylline do not. By contrast to caffeine, CB002-analogues target an Sphase checkpoint associated with increased p-RPA/RPA2, p-ATR, decreased Cyclin A, p-histone H3 expression and downregulation of essential proteins in DNA-synthesis and -repair. CB002-analogue #4 enhances cell death, and decreases Ki-67 in patient-derived tumor-organoids without toxicity to normal human cells. Preliminary in vivo studies demonstrate anti-tumor efficacy in mice. Thus, a novel class of anti-cancer drugs show activation of p53 pathway signaling in tumors with mutated p53, and target an S-phase checkpoint.

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Abstract C189: Defective S phase cell cycle checkpoint: A potential culprit and target in melanoma

10.1158/1535-7163.targ-15-c189 ◽

2015 ◽

Author(s):

Zay Y. Oo ◽

Sheena Daignault ◽

James Chen ◽

Brian Gabrielli

Keyword(s):

Cell Cycle ◽

S Phase ◽

Cell Cycle Checkpoint ◽

Phase Cell ◽

Cycle Checkpoint

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The Wee1 Inhibitor MK1775 In Combination With Cytarabine (AraC) Has Potent Activity In AML By Completely Abrogating DNA Damage and Cell Cycle Checkpoint Repair Capacity Via The Mrn/NBS1 Complex

Blood ◽

10.1182/blood.v122.21.3831.3831 ◽

2013 ◽

Vol 122 (21) ◽

pp. 3831-3831

Author(s):

Leena Chaudhuri ◽

James M Bogenberger ◽

Lisa Sproat ◽

James L Slack ◽

Veena Fauble ◽

...

Keyword(s):

Cell Cycle ◽

Dna Damage ◽

Single Agent ◽

S Phase ◽

Cell Cycle Checkpoint ◽

Leukemic Cells ◽

Repair Capacity ◽

Strand Breaks ◽

Mitotic Entry ◽

Cycle Checkpoint

Abstract Cytarabine (AraC) resistance is a fundamental feature of refractory/relapsed AML. RNA interference (RNAi) screens conducted in our laboratory recently identified WEE1 kinase (WEE1) as one of the top candidate genes and target in leukemias in combination with AraC. WEE1 is a tyrosine kinase belonging to the Ser/Thr family of protein kinases and acts as a negative regulator of mitotic entry by controlling DNA damage (DDR) and cell cycle checkpoint responses. The WEE1 inhibitor MK1775 potently synergizes with AraC ex vivo and in vitro and clinical trials are in preparation. However, the mechanism of action for the anti-leukemic activity of MK1775 with AraC remains unknown. To elucidate genes mediating activity of the combination, we first performed siRNA rescue screens silencing a custom set of 44 genes involved in WEE1 regulation under combined AraC + MK1775 to identify sensitizers and markers of resistance. The MRN (MRE11, Rad51, NBS1) complex and particularly NBS1 were potent modifiers of AraC and MK1775. Focusing on NBS1 since it is proposed to centrally regulate the defense capacity of leukemic cells, we identified that NBS1 phosphorylation at Ser343 (the ATM regulation site) is significantly altered both in cell lines and primary AML samples under combined AraC+MK1775 treatment as compared to single agent MK1775. In parallel, lower phosphorylation of ATMS1981(an autophosphorylation site in response to DNA strand breaks), was observed indicating that the ATM-CHEK1 pathway is not activated under co-treatment. Further Homologous recombination (HR)-mediated repair was compromised by AraC+MK1775 shown by DR-GFP expression vector to measure intracellular HR capacity: post-transfection of the I-SceI nuclease which cleaves non-functioning GFP tandem repeats to form a functional GFP unit, the HR was reduced with the combination. Consistently other HR markers decreased as well. Delayed accumulation of Cyclin A (indicative of S-phase progression) and greater inhibition of phospho-Cdk2Y15in synchronized cells treated with AraC + MK1775 in comparison to controls was observed. In addition the cell cycle was globally dysregulated by slower S-phase kinetics (progression), a completely abrogated G2/M checkpoint/phase as well as de-regulated DNA replication origin formation and firing as evidenced by Cdt1 and Mus81. As a consequence high single and double strand breaks (ɣH2AX) were observed with an increase in phospho-histone H3 in AraC + MK1775 treated cells compared to untreated cells or MK1775 single agent, confirming faster mitotic entry. Changes were followed by massive induction of apoptosis. Since WEE1 is implicated in leukemic stem cell maintenance we examined the long term effects of the combination in colony forming assays. AraC + MK1775 treated leukemic cells obtained from patients with AML were re-plated on Methocult after drug washout and colonies counted after 14 days. While MK1775 as a single agent could reduce colony formation by 4 fold compared to controls and lower dose AraC, co-treatment with low to moderate doses of AraC and MK1775 reduced colony formation by more than 7 fold and to almost zero in some primary specimens. Taken together, these results suggest that leukemia cells co-treated with AraC + MK1775 lost their ability to activate DNA damage and repair pathways mainly by compromising the MRN complex via NBS1 with subsequently reduced HR. The combination (as opposed to single agents) almost complete dysregulated the cell cycle and its checkpoints lead to DNA damage, genomic instability and rapid exit from the cell cycle with cell death via apoptosis. Thus we have molecularly characterized the detailed mechanisms underlying the potent AraC+WEE1 inhibition in AML and describe for the first time a therapeutic combination that has the potential to abrogate the MRN and NBS1 repair capacity which is central for drug resistance in AML. A key implication of our work is to provide a clinical rationale, mechanistic understanding and suggestions for biomarkers to clinically evaluate AraC + MK1775 in patients with AML. Disclosures: No relevant conflicts of interest to declare.

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47 Preclinical studies of dianhydrogalactitol (VAL-083) in DIPG, as single agent or as a combination with AZD1775 or radiation

Canadian Journal of Neurological Sciences / Journal Canadien des Sciences Neurologiques ◽

10.1017/cjn.2018.286 ◽

2018 ◽

Vol 45 (S3) ◽

pp. S9-S10

Author(s):

Xiaodong Yang ◽

Anne Steino ◽

Jeffrey Bacha ◽

Dennis Brown ◽

Sabine Mueller

Keyword(s):

Cell Cycle ◽

Cell Lines ◽

Dna Methyltransferase ◽

Cell Cycle Progression ◽

Single Agent ◽

Chemotherapeutic Agents ◽

Phase Cell ◽

Dna Double Strand Breaks

Despite decades of trials, the prognosis for diffuse intrinsic pontine gliomas (DIPG) remains dismal. DIPG is inoperable and standard treatment is radiation alone, as the addition of chemotherapeutic agents, such as temozolomide, have not improved survival. In addition to inherent chemoresistance, treatment of DIPG is impeded by an intact blood-brain barrier (BBB). VAL-083 is a structurally unique bi-functional DNA-targeting agent that readily crosses the BBB. VAL-083 forms interstrand DNA crosslinks at N7-guanine, resulting in DNA double-strand breaks (DSB), S/G2-phase cell-cycle arrest, and ultimately cancer cell death. We have previously demonstrated that VAL-083 is able to overcome temozolomide-resistance in vitro and in vivo, and that its cytotoxicity is independent of the DNA-repair enzyme O6-methylguanine DNA-methyltransferase (MGMT). MGMT is almost universally expressed in DIPG and its expression is strongly correlated with temozolomide-resistance. VAL-083’s distinct mechanism-of-action suggests the potential for combination with inhibitors of DNA DSB repair or S/G2 cell-cycle progression (e.g. Wee1 inhibitor AZD1775). Here, we investigated the effects of VAL-083 in combination with radiation, AZD1775 or irinotecan (topoisomerase inhibitor) in three DIPG cell-lines: SF10693 (H3.1), SF8628 (H3.3) and NEM157 (H3.3). VAL-083 showed activity at low uM-concentration in all three cell-lines. In addition, VAL-083 showed synergy with AZD1775 in all three cell-lines. Combined with its ability to cross the BBB, accumulate in brain tumor tissue and overcome MGMT-related chemoresistance, these results suggest VAL-083 as a potentially attractive treatment option for DIPG as single agent or in combination with AZD1775. Combination studies with radiation are ongoing and will be presented at the meeting.

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Physiological electric fields control the G 1 /S phase cell cycle checkpoint to inhibit endothelial cell proliferation

The FASEB Journal ◽

10.1096/fj.02-0510fje ◽

2003 ◽

Vol 17 (3) ◽

pp. 1-14 ◽

Cited By ~ 37

Author(s):

Entong Wang ◽

Yili Yin ◽

Min Zhao ◽

John V. Forrester ◽

Colin D. McCaig

Keyword(s):

Cell Cycle ◽

Cell Proliferation ◽

Endothelial Cell ◽

Electric Fields ◽

S Phase ◽

Cell Cycle Checkpoint ◽

Phase Cell ◽

Endothelial Cell Proliferation ◽

Cycle Checkpoint

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Imaging with FLT-PET demonstrates that PF-477736, an inhibitor of CHK1 kinase, overcomes a cell cycle checkpoint induced by gemcitabine in PC-3 xenografts

Journal of Clinical Oncology ◽

10.1200/jco.2006.24.18_suppl.3045 ◽

2006 ◽

Vol 24 (18_suppl) ◽

pp. 3045-3045 ◽

Cited By ~ 5

Author(s):

G. A. McArthur ◽

J. Raleigh ◽

A. Blasina ◽

C. Cullinane ◽

D. Dorow ◽

...

Keyword(s):

Cell Cycle ◽

Dna Damage ◽

Fold Increase ◽

Cell Cycle Checkpoint ◽

Flt Pet ◽

Cycle Arrest ◽

Cycle Checkpoint ◽

A Cell ◽

Chk1 Inhibitor

3045 Background: The development of strategies to monitor the molecular and cellular response to novel agents that target the cell cycle is vital to provide proof of mechanism and biological activity of these compounds. The protein kinase CHK1 is activated following DNA damage in the S and G2-phases of the cell cycle and mediates cell cycle arrest. In vitro studies demonstrate that inhibition of CHK1 can overcome cell cycle arrest induced by DNA damage and enhance cytotoxic activity of DNA damaging agents. In vivo studies show that combining DNA damaging agents with a CHK1 inhibitor potentiates antitumor activity. We hypothesize that functional imaging with 18F-fluorine-L-thymidine (FLT), a PET-tracer where tumor uptake is maximal in the S and G2 phases of the cell cycle can be used to non-invasively monitor the induction and therapeutic inhibition of a cell cycle checkpoint in vivo. Methods: Nude mice harbouring PC-3 xenografts were treated with vehicle controls, gemcitabine, the CHK1-inhibitor PF-477736 or gemcitabine + PF-477736. FLT-PET scans were performed and tumors harvested for ex-vivo biomarkers to assess S-phase, M-phase and DNA-repair. Results: Gemcitabine induced a 8.3 ±0.8 fold increase in tumoral uptake of FLT at 21 hours that correlated with a 3.3 ±0.2-fold increase in thymidine kinase activity and S-phase arrest as demonstrated by BrdU incorporation and elevated expression of cyclin-A. Treatment with PF-477736 at 17 hours after gemcitabine abrogated the early FLT-flare at 21 hours by 82% (p<0.001). This was associated with both an increased fraction of cells in mitosis and G1-phase of the cell cycle as determined by phos-histone H3 and flow cytometry. Furthermore, the combination of gemcitabine and PF-477736 enhanced DNA damage as measured by phos-gamma-H2AX and significantly delayed tumor growth when compared to tumors treated with gemcitabine alone. Conclusion: These data clearly indicate that the CHK1-inhibitor PF-477736 can overcome the cell cycle checkpoint induced by gemcitabine and increase associated DNA damage in tumors in-vivo. The PET studies indicate that functional imaging with FLT-PET is a promising strategy to monitor responses to therapeutic agents that target cell cycle checkpoints. [Table: see text]

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